Silk particles for controlled and sustained delivery of compounds

ABSTRACT

The present invention relates to a method of producing and loading silk particles, preferably spider silk particles, with a compound. In particular, the present invention provides a novel two step method for loading silk particles, preferably spider silk particles, with small and water-soluble compounds. Also disclosed are silk particles, preferably spider silk particles, loaded with at least one compound which are eminently suited as carriers for controlled and sustained delivery applications. Furthermore, the invention relates to pharmaceutical or cosmetic compositions comprising said silk particles, preferably spider silk particles, and a pharmaceutically active compound or cosmetic compound for controlled and sustained release. The present invention is also directed to silk particles, preferably spider silk particles, loaded with a compound obtainable by the method according to the invention.

This application is a National Stage of International Application No.PCT/EP2010/007266, filed Nov. 30, 2010, and entitled SILK PARTICLES FORCONTROLLED AND SUSTAINED DELIVERY OF COMPOUNDS, which is incorporatedherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of producing and loading silkparticles, preferably spider silk particles, with a compound. Inparticular, the present invention provides a novel two step method forloading silk particles, preferably spider silk particles, with small andwater-soluble compounds. Also disclosed are silk particles, preferablyspider silk particles, loaded with at least one compound which areeminently suited as carriers for controlled and sustained deliveryapplications. Furthermore, the invention relates to pharmaceutical orcosmetic compositions comprising said silk particles, preferably spidersilk particles, and a pharmaceutically active compound or cosmeticcompound for controlled and sustained release. The present invention isalso directed to silk particles, preferably spider silk particles,loaded with a compound obtainable by the method according to theinvention.

BACKGROUND

In the past years sophisticated drug depot systems for controlleddelivery of substances have been developed, for example to achieveconstant drug levels in plasma during therapy. These systems have theadvantage of reducing toxic side effects so that the number of drugadministrations can be decreased, while at the same time improvingcellular uptake and bioavailability. Especially colloidal micro- andnanoparticulate carriers have been extensively investigated as aplatform for controlled drug delivery. There is also an ongoing quest todesign nano- or microparticles which facilitate controlled release ofsubstances other than pharmaceutical compounds. In general, the materialemployed as carrier for controlled and sustained release of a substanceshould offer control of structure, morphology and function, while alsoexhibiting good mechanical stability.

For example, biodegradable and biocompatible polymers are preferredbecause of their ability to retain their properties for a limited periodof time before gradually decomposing into soluble nontoxic degradationproducts which can be excreted from the body. Many synthetic (aliphaticpolyesters, polyglycolic acid (PGA), polylactid acid (PLA), etc.) andnatural (polysaccharides, chitin, chitosan, proteins) polymers have beenemployed to produce degradable vehicles for encapsulation, incorporationor binding of active compounds [Freiberg, S., Zhu, X. X. Polymermicrospheres for controlled drug release. International Journal ofPharmaceutics 2004; 282(1-2):1-18].

While synthetic polymers potentially posses the feature of sustainedrelease of the encapsulated therapeutic agent from a period of days upto several months, they typically demand organic solvents or relativelyharsh formulation conditions during processing with potentially limitedbiocompatibility because of remaining toxic solvents and acidicdegradation products.

A further advance in the art was to consider natural polymers which havethe advantage of being biocompatible. However, most biopolymers known atpresent have a major drawback, namely that they resolubilize rapidly inaqueous environment due to their hydrophilic nature, thus resulting infast drug release profiles. In order to circumvent this problem,chemical cross-linking procedures have been considered. Unfortunately,the presence of residual cross-linking agents can lead to toxic sideeffects. In addition, undesirable reactions between the drug and thecross-linker could result in the formation of either toxic orinactivated derivatives.

The use of hydrophobic biopolymers as carriers for sustained drugrelease has also been investigated in the art. For example, silkproteins have been considered as being suitable biopolymers. Inparticular, silk proteins from spiders and insects, especially Bombyxmori fibroin, have been tested for their ability to deliver drugs andother substances.

For example, silk microspheres consisting of silkworm fibroin forencapsulation and controlled release of a model protein drug has beendescribed in the art. These silk fibroin microspheres with diameters ofseveral microns are obtained by a method using lipid vesicles as atemplate [Wang, X., Silk microspheres for encapsulation and controlledrelease. Journal of Controlled Release 2007; 117(3): 360-370].

Larger silk fibroin particles with diameters ranging from 100 to 440 μmand improved loading efficiencies have also been described in the art.However, the preparation techniques for producing these particles arehighly sophisticated and lack scalability [Wenk, E., Silk fibroinspheres as a platform for controlled drug delivery. Journal ofcontrolled release 2008; 132(1):26-34].

WO 2007/014755 describes a method of producing nano- and microscapsulesconsisting of spider silk proteins. These capsules with sizes of severalmicrons are composed of an outer spider silk protein shell and cangenerally be filled with substances such as proteins or chemicalreactants. The microcapsules are formed by the encapsulation of emulsiondroplets resulting in hollow spider silk protein shells.

WO 2007/0829223 relates to the use of protein microbeads in cosmetics.In particular, this international patent application describes proteinmicrobeads composed of synthetic spider silk proteins for delivery ofcosmetic substances [Hümmerich, D., Primary structure elements of spiderdragline silks and their contribution to protein solubility.Biochemistry 2004 Oct. 26; 43(42): 13604-13612]. Similarly, WO2007/082923 describes the use of protein microbeads for formulatingpoorly water-soluble effect substances. In both patent applications, thewater-insoluble effect substances can be either associated with orencapsulated in the protein microbeads. The association of thesubstances to these beads is mainly due to hydrophobic interactions.This encapsulation strategy has the basic disadvantage that the loadedsubstances are only released upon proteolysis of the protein microbeadsby the activity of proteases which makes a constant and controlledrelease difficult. A further problem is that this system is onlysuitable for the formulation of mainly water-insoluble substances.

Hence, there is a strong need in the field to provide a novel method ofproducing micro- or submicroparticles with improved qualities. Inparticular, there is still an ongoing quest to produce nano-scaledparticles which are biocompatible and biodegradable as well as beingstable carriers for small and water soluble compounds. There is also aneed to provide a suitable method of loading silk particles, e.g. spidersilk particles, effectively with a compound of interest. The silkparticles, e.g. spider silk particles, should also be capable ofreleasing the loaded compound controllably and sustainably.

Accordingly, it is an object of the present invention to provide a noveland simple drug delivery system which takes into account all of theabove criteria. The present invention, therefore, provides a novelmethod of producing silk particles, preferably spider silk particles,loaded with a compound. More particularly, the method comprises thesteps of providing silk particles, preferably spider silk particles,comprising one or more silk polypeptides, preferably spider silkpolypeptides, comprising at least two identical repetitive units, andincubating said silk particles, preferably spider silk particles, withat least one compound, wherein the compound is water-soluble and has amolecular weight of between about 50 Da and about 20 kDa.

Surprisingly, one major advantage of the silk carrier system accordingto the invention is that these particles can be produced and loadedwithin an all-aqueous system and under ambient condition. This isparticularly important with regard to the loading of labile compounds aswell as to the overall biocompability of the product. The silkparticles, e.g. spider silk particles, of the invention have revealedunexpected loading efficiencies for small and water-soluble compounds.Surprisingly, the silk particles, e.g. spider silk particles, obtainedby the method according to the invention have further demonstrated amost favourable release profile, rendering them eminently suitable forcontrolled and sustained delivery of a compound. The produced silkparticles, e.g. spider silk particles, are, therefore, very well suitedfor delivery of pharmaceutical and cosmetic compounds. Due to theircolloidal stability and biocompability under physiological conditions,the loaded silk particles, e.g. spider silk particles, according to theinvention are especially suitable for in vivo applications.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method of producing silkparticles, preferably spider silk particles, loaded with a compoundcomprising the steps of

-   -   i) providing silk particles, preferably spider silk particles,        comprising one or more silk polypeptides, preferably spider silk        polypeptides, comprising at least two identical repetitive        units, and    -   ii) incubating said silk particles, preferably spider silk        particles, with at least one compound,        -   wherein the compound is water-soluble and has a molecular            weight of between about 50 Da and about 20 kDa.

In a preferred embodiment of the invention, the compound has a molecularweight of 50 Da or about 50 Da to 10 kDa or about 10 kDa, preferably 50Da or about 50 Da to 6 kDa or about 6 kDa, more preferably 50 Da orabout 50 Da to 4 kDa or about 4 kDa and most preferably 50 Da or about50 Da to 1 kDa or about 1 kDa.

In preferred embodiments of the invention, the silk particles,preferably spider silk particles, provided in step i) are produced bythe steps of

-   -   a) providing an aqueous solution comprising one or more silk        polypeptides, preferably spider silk polypeptides, comprising at        least two identical repetitive units,    -   b) triggering aggregation of the silk polypeptides, preferably        spider silk polypeptides, to form silk particles, preferably        spider silk particles, and    -   c) separating the silk particles, preferably spider silk        particles, by phase separation.

Preferably, the compound is able to permeate into the matrix of the silkparticles, preferably spider silk particles.

In further preferred embodiments, at least 40%, preferably at least 50%,60%, 70%, 80%, 90%, or 95% of the loaded compound is located within thematrix of the silk particles, preferably spider silk particles.

In further preferred embodiments, the silk particles, preferably spidersilk particles, have a median size of between 0.1 μm and 500 μm,preferably of between 0.1 μm and 100 μm, more preferably of between 0.2μm and 20 μm, even more preferably of between 0.2 to 1 μm, and mostpreferably of between 0.25 μm and 0.7 μm.

In preferred embodiments, the at least two identical repetitive unitseach comprise at least one consensus sequence selected from the groupconsisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from the group consisting of        A, S, G, Y, P and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from the group consisting of Y, P, R, S,        A, T, N and Q; and    -   iii) A_(x), wherein x is an integer from 5 to 10.

In further preferred embodiments, the repetitive unit(s) of therespective silk polypeptide, preferably spider silk polypeptide, is(are) independently selected from module A (SEQ ID NO: 20) or variantsthereof, module C (SEQ ID NO: 21) or variants thereof, module Q (SEQ IDNO: 22) or variants thereof, module A^(C) (SEQ ID NO: 29), module A^(K)(SEQ ID NO: 30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO:32), module C^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).

In further specific embodiments, the silk polypeptide, preferably spidersilk polypeptide, further comprises at least one non-repetitive (NR)unit.

More preferably, the non-repetitive (NR) unit is independently selectedfrom the group consisting of NR3 (SEQ ID NO: 41 and SEQ ID NO: 45) orvariants thereof and NR4 (SEQ ID NO: 42 and SEQ ID NO: 46) or variantsthereof.

In further specific embodiments, the silk polypeptide, preferably thespider silk polypeptide, is selected from the group consisting of ADF-3(SEQ ID NO: 1 and SEQ ID NO: 47), ADF-4 (SEQ ID NO: 2 and SEQ ID NO:48), MaSp I (SEQ ID NO: 43 and SEQ ID NOs: 53-64), MaSp II (SEQ ID NO:44 and SEQ ID NOs: 65-78), (C)_(m)NR_(z), NR_(z)(C)_(m), (AQ)_(n)NR_(z),NR_(z)(AQ)_(n), NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z), (C)_(m), (AQ)_(n), and(QAQ)_(o), wherein m is an integer of 8 to 48, n is an integer of 6 to24, o is an integer of 8 to 16, z is an integer of 1 to 3.

More preferably, the silk polypeptide, preferably spider silkpolypeptide, is C₁₆, C₃₂, (AQ)₁₂, (AQ)₂₄, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3, or(AQ)₂₄NR3.

In further preferred embodiments of the invention, the concentration ofthe silk polypeptide, preferably spider silk polypeptide, in the aqueoussolution is of between 0.01 wt %/vol and 30 wt %/vol, more preferablybetween 0.1 wt %/vol and 30 wt %/vol, and most preferably between 1 wt%/vol and 20 wt %/vol.

In further specific embodiments, the aggregation is triggered by pHshift, ion exchange, shear forces, the addition of alcohol, or alyotropic salt or by combinations thereof. More preferably the alcoholis methanol.

Also preferably, the lyotropic salt is selected from the groupconsisting of ammonium sulphate, sodium phosphate, and potassiumphosphate.

More preferably, the concentration of the lyotropic salt is of betweenabout 400 mM and about 3 M, preferably about 1 to about 2 M, mostpreferably about 2 M.

In preferred embodiments of the invention, the compound is apharmaceutically active compound, a cosmetic substance, an agriculturalsubstance, a chemoattractant, a chemorepellent, an anti-fungalsubstance, an anti-bacterial substance, a nutrient, a dietarysupplement, a dye, a fragrance or an agent selected from the groupconsisting of hemostatic agents, growth stimulating agents, inflammatoryagents, anti-fouling agents, antimicrobial agents and UV protectingagents.

In further specific embodiments, the compound has an overall positivenet charge.

In further specific embodiments, the compound is able to permeate intothe silk matrix, preferably spider silk matrix, by electrostaticinteraction and/or diffusion. In preferred embodiments, the compound hasa neutral or alkaline nature. In further specific embodiments, step ii)of the method is carried out at temperatures of between 4° C. and 40°C., preferably of between 10° C. and 30° C. and more preferably ofbetween 20° C. and 25° C.

In further specific embodiments, step ii) of the method is carried outat a pH of between 1 and 9, preferably of between 4 and 9 and mostpreferably of between 6 and 8.

In a second aspect, the present invention relates to silk particles,preferably spider silk particles, comprising at least one silkpolypeptide, preferably spider silk polypeptide, comprising at least twoidentical repetitive units loaded with at least one compound, which iswater-soluble and has a molecular weight of between about 50 Da andabout 20 kDa.

In a preferred embodiment of the invention, the compound has a molecularweight of 50 Da or about 50 Da to 10 kDa or about 10 kDa, preferably 50Da or about 50 Da to 6 kDa or about 6 kDa, more preferably 50 Da orabout 50 Da to 4 kDa or about 4 kDa and most preferably 50 Da or about50 Da to 1 kDa or about 1 kDa.

In further preferred embodiments, at least 40%, preferably 50%, 60%,70%, 80%, 90%, or 95% of the loaded compound is located within thematrix of the silk particles, preferably spider silk particles.

In preferred embodiments of the invention, the median size of theparticles is 0.1 μm to 500 μm, preferably 0.1 μm to 100 μm, morepreferably 0.2 μm to 20 μm, even more preferably 0.2 μm to 1 μm and mostpreferably 0.25 μm to 0.7 μm.

In further specific embodiments, the at least two identical repetitiveunits each comprise at least one consensus sequence selected from thegroup consisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from the group consisting of        A, S, G, Y, P and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from the group consisting of Y, P, R, S,        A, T, N and Q; and    -   iii) A_(x), wherein x is an integer from 5 to 10.

More preferably, the repetitive unit(s) of the silk polypeptide,preferably spider silk polypeptide, is (are) independently selected frommodule A (SEQ ID NO: 20) or variants thereof, module C (SEQ ID NO: 21)or variants thereof, module Q (SEQ ID NO: 22) or variants thereof,module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO: 30), module C^(C)(SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), module C^(K2) (SEQ IDNO: 33) or module C^(KC) (SEQ ID NO: 34).

In further specific embodiments, the silk polypeptide, preferably spidersilk polypeptide, further comprises one or more non-repetitive (NR)units.

More preferably, the NR unit is independently selected from the groupconsisting of NR3 (SEQ ID NO: 41 and SEQ ID NO: 45) or variants thereofand NR4 (SEQ ID NO: 42 and SEQ ID NO: 46) or variants thereof.

In preferred embodiments of the invention, the silk polypeptide,preferably spider silk polypeptide, is selected from the groupconsisting of ADF-3 (SEQ ID NO: 1 and SEQ ID NO: 47), ADF-4 (SEQ ID NO:2 and SEQ ID NO: 48), MaSp I (SEQ ID NO: 43 and SEQ ID NOs: 53-64), MaSpII (SEQ ID NO: 44 and SEQ ID NOs: 65-78), (C)_(m)NR_(z), NR_(z)(C)_(m),(AQ)_(n)NR_(z), NR_(z)(AQ)_(n), NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z),(C)_(m), (AQ)_(n), and (QAQ)_(o), wherein m is an integer of 8 to 48, nis an integer of 6 to 24, o is an integer of 8 to 16, z is an integer of1 to 3.

More preferably, the silk polypeptide, preferably spider silkpolypeptide, is C₁₆, C₃₂, (AQ)₁₂, (AQ)₂₄, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3, or(AQ)₂₄NR3.

In further preferred embodiments of the invention, the compound is apharmaceutically active compound, a cosmetic substance, an agriculturalsubstance, a chemoattractant, a chemorepellent, an anti-fungalsubstance, an anti-bacterial substance, a nutrient, a dietarysupplement, a dye, a fragrance or an agent selected from the groupconsisting of hemostatic agents, growth stimulating agents, inflammatoryagents, anti-fouling agents, antimicrobial agents and UV protectingagents.

In further specific embodiments, the compound has an overall positivenet charge.

In further specific embodiments, the compound is able to permeate intothe silk matrix, preferably spider silk matrix, by electrostaticinteraction and/or diffusion.

In further preferred embodiments, the compound has a neutral or alkalinenature.

In preferred embodiments of the invention, the compound is released fromthe silk particles, preferably spider silk particles, by diffusion uponexposure to physiological conditions.

In further preferred embodiments, less than 20%, preferably less than15%, and most preferably less than 10% of the compound is releasedwithin the first 24 hours.

In a third aspect, the invention relates to a pharmaceutical compositioncomprising the silk particles; preferably spider silk particles,according to the invention and additionally a pharmaceuticallyacceptable buffer, diluent and/or excipient for controlled and sustaineddelivery, wherein the compound is a pharmaceutically active compound.

In a fourth aspect, the invention relates to a cosmetic compositioncomprising the silk particles, preferably spider silk particles,according to the invention for controlled and sustained delivery,wherein the compound is a cosmetic compound.

In a fifth aspect, the invention relates to silk particles, preferablyspider silk particles, loaded with a compound, wherein the compound iswater soluble, has a molecular weight of about 50 Da to about 20 kDa andhas an overall positive net charge and wherein the silk particles,preferably spider silk particles, comprise one or more silkpolypeptides, preferably spider silk polypeptides, comprising at leasttwo identical repetitive units, the particles being obtainable by aprocess according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: eADF4(C16) particle characterization: a) Size distribution ofobtained eADF4(C16) particles analyzed using laser diffractionspectrometry. The inset, shows an scanning electron micrograph ofcorresponding eADF4(C16) particles. The average diameter of the particleensemble was d_(avg)=332±95 nm. b) Investigation of colloidal stabilityassessed by intensity of scattered light at 400 nm. R² is thecorrelation coefficient of the linear fit.

FIG. 2: Characterization of loading procedure: a) Zeta-Potential ofeADF4(C16) particles as a function of added methyl violet. Forcomparison, the inlay shows the Zeta-potential of glass beads withmethyl violet. b) Loading and loading efficiency of methyl violet oneADF4(C16) particles as a function of molar ratio.

FIG. 3: Loading efficiencies for model drugs of weak alkaline naturesuch as Ephedrin (Eph), Procain (Prc), Propranolol (Prp), Papaverine(Pap) and Tetracaine (Tet) plotted over log D MW⁻¹.

FIG. 4: Release studies of ethacridine lactate and methyl violet: a)Experimental and theoretical release kinetics of both model drugs over aperiod of 35 days. b) Experimental and theoretical release kinetics inthe initial burst region (release <11%). c) Release of ethacridinelactate as a function of pH as indicated. (Buffer capacity PBS: pH5.8-pH 8; non buffered conditions for pH 2.0, pH 4.0 and pH 8.8) d)Experimental release data of ethacridine lactate and methyl violet basedon the power law model. A linear fit with a correlation parameter (r²)above 0.99 was determined for three distinct time intervals. The linearfit for the interval from day 1 to day 13 is depicted in the main plot,whereas the inset shows the data and linear fits for the time intervalsfrom day 14 to day 20 (open symbols) and day 21 to day 35 (filledsymbols) respectively.

FIG. 5: Characterization of eADF4(C16) particles upon enzymaticdegradation: a) Size distribution of eADF4(C16) particles upon enzymaticdegradation at time points as indicated. b) Mean and mode of eADF4(C16)particles distribution over time. c) Percentage of particles andagglomerations of eADF4(C16) particles after degradation with elastase(c=4 μg/ml) and trypsin (c=50 μg/ml) at timepoints as indicated. d)Second derivative of FTIR spectra of eADF4(C16) particles upondegradation at time points as indicated.

FIG. 6: A) Loading and loading efficiencies of lysozyme on C₁₆ spidersilk particles as a function of w/w-ratio at pH 7.0/30 mM. The loadingefficiency ranges above 90% for w/w ratios up to 30%, representing avery effective loading process (more than 90% of the overall addedlysozyme is bound to/permeated into the particle). At w/w rations above30% the loading efficiency slowly decreases, resulting in higher amountsof unloaded lysozyme in solution. B) Zeta-potential of spider silkparticles after loading with different amounts of lysozyme at pH 7.0/30mM.

FIG. 7: Loading of lysozyme onto C₁₆ spider silk particles at differentionic strength at pH 7.0. A) Loading of lysozyme as a function ofw/w-ratio lysozyme to spider silk particles. B) Loading efficiencies oflysozyme as a function of w/w-ratio lysozyme to spider silk particles.

FIG. 8: Particle size of C₁₆ spider silk particles loaded with differentw/w ratios of lysozyme to spider silk particles. The size of the spidersilk particles loaded with approximately 10% [w/w] lysozyme did notdiffer from unloaded spider silk particles. “Pdi” means polydispersityindex.

DETAILED DESCRIPTION OF THE INVENTION

It is a primary object of the present invention to find a simple, mildand efficient way of producing silk particles, e.g. spider silkparticles, with improved qualities for controlled and sustained deliveryof a compound. It is another object to provide a novel and simple twostep method for loading of silk particles, e.g. spider silk particles,with a compound of interest, thereby circumventing the disadvantages anddrawbacks of the conventional methods of loading silk particles, e.g.spider silk particles, known from the art. In particular, it is yetanother object to provide a method for loading of small andwater-soluble compounds effectively. Another object of the invention isto provide silk particles, e.g. spider silk particles, having favourablecarrier characteristics. One major advantage is that the particlesproduced by the method of the present invention are small in size,colloidally stable, biocompatible as well as biodegradable, and show anoverall constant release profile. Other objects and advantages of thepresent invention will be apparent from the further reading of thespecification and of the appended claims.

The present invention has solved the problems of the prior art byconsidering and making use of the intrinsic properties of the silkprotein, e.g. spider silk protein, as well as of the compound to beloaded. Surprisingly, the inventors discovered that especially small andwater-soluble compounds can be effectively loaded onto the silkparticles, e.g. spider silk particles, under very mild conditions. Itwas further an unexpected finding that the method according to theinvention can be conducted without using any organic solvents or toxiccross-linking chemicals, thereby avoiding relatively harsh formulationconditions. In particular, it was quite surprising that the methodaccording to the invention can be carried out in an all-aqueous process.One major advantage of the method according to the present invention isthat the particles are produced in a first step and are afterwardsloaded with a compound of interest in a second step. Thus, contrary tothe methods of the art, said two steps of the method according to theinvention can be carried out separately, i.e. both spatially as well asat different times. Further, it was also surprising that the loadedcompound can be continuously and controllably released once produced,which renders the silk particles, e.g. spider silk particles, accordingto the invention a very suitable carrier system, especially wheresustained delivery of a compound is required. Because of their goodbiocompatibility as well as biodegradability, these silk particles, e.g.spider silk particles, are eminently suitable in pharmaceutical andcosmetic applications. It is however also evident that the silkparticles, e.g. spider silk particles, according to the invention arenot only limited to medical and cosmetic use. Depending on the nature ofthe loaded compound, the silk particles, e.g. spider silk particles,produced by the method according to the invention can also be employedas a carrier system for practically any kind of substances, e.g.nutrients, dietary supplements, dyes, fragrances, and a variety of otheragents.

Some of the used terms will hereinafter be defined in greater detailbelow: Where the term “comprising” is used in the present descriptionand the claims, it does not exclude other elements or steps. For thepurposes of the present invention, the term “consisting of” isconsidered to be a preferred embodiment of the term “comprising”. Ifhereinafter a group is defined as comprising at least a certain numberof embodiments, this is also to be understood as disclosing a groupwhich preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to asingular noun e.g. “a”, “an” or “the”, this includes a plural of thatnoun unless something else is specifically stated.

The term “about” in the context of the present invention denotes aninterval of accuracy that the person skilled in the art will understandto still ensure the technical effect of the feature in question. Theterm typically indicates deviation from the indicated numerical value of±10%, preferably 5%, most preferably 2%.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. It is well known in the art that analogous positions in twoor more polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5.

Concentrations, amounts, solubilities, and other numerical data may beexpressed or presented herein in a range format. It is to be understoodthat such a range format is used merely for convenience and brevity andthus should be interpreted flexibly to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. As an illustration, a numerical range of “about 1 to about 5”should be interpreted as including not only the explicitly recitedvalues of about 1 to about 5, but also include individual values andsub-ranges within the indicated range. Thus, included in this numericalrange are individual values such as 2, 3, and 4 and sub-ranges such asfrom 1-3, from 2-4 and from 3-5, etc. This same principle applies toranges reciting only one numerical value. Furthermore, such aninterpretation should apply regardless of the breadth of the range orthe characteristic being described.

Within the context of the present invention, “median size” or “meansize” or “median particle size” or “mean particle size” can be usedinterchangeably and define the median silk particle size, preferablyspider silk particle size, i.e., the silk particle, preferably spidersilk particle diameter, where 50% of the silk particles, preferablyspider silk particles, are smaller and 50% of the silk particles,preferably spider silk particles, are larger than the stated value.Usually, this corresponds to the maximum of a Gaussian sizedistribution.

The present invention will hereinafter be described with respect toparticular embodiments and with reference to certain drawings, althoughthe invention is not limited thereto, but only by the claims.

In a first aspect, the invention relates to a method of producing silkparticles, preferably spider silk particles, loaded with a compoundcomprising, essentially consisting of, or consisting of the steps of:

-   -   i) providing silk particles, preferably spider silk particles,        comprising one or more silk polypeptides, preferably spider silk        polypeptides, comprising at least two identical repetitive        units, and    -   ii) incubating said silk particles, preferably spider silk        particles, with at least one compound,        -   wherein preferably the compound is water-soluble and/or has            a molecular weight of between about 50 Da and about 20 kDa.

It is to be understood that the method according to the invention is aprocess of two or more steps, wherein the particles are produced in afirst step and are afterwards loaded with a compound of interest in asecond step. Thus, contrary to the methods of the art, said, at leasttwo steps of the method according to the invention can be carried outseparately, i.e. both spatially as well as at different times. Accordingto preferred embodiments of the present invention, the steps i) and ii)of the method are carried out in separated processes, i.e. step ii)follows the provision of the silk particles, e.g. spider silk particles,in step i). Surprisingly, in contrast to the state of the art (e.g. inthe form of WO 2007/082923), the second step in this invention is mainlybased of diffusion of a compound into the matrix of the silk particle,e.g. spider silk particle, leading to a highly efficient permeation.

As used herein, “silks”, e.g. “spider silks”, are protein polymers thatdisplay extraordinary physical properties. Among the different types ofsilks, e.g. spider silks, draglines are most intensely studied. Draglinesilks are generally utilized by orb weaving spiders to build frame andradii of their nets and as lifelines that are permanently draggedbehind. For these purposes, high tensile strength and elasticity arerequired. The combination of such properties results in a toughness thatis greater than that of most other known materials.

Dragline silks are generally composed of two major proteins whoseprimary structures share a common repetitive architecture. For example,the two major protein components of draglines from Nephila clavipes aretermed MaSp1 and MaSp2 (Major ampullate Spidroins) and from Araneusdiadematus ADF-3 and ADF-4 (Araneus Diadematus Fibroin). The draglinesilk proteins have apparent molecular masses between 180 kDa and 720 kDadepending on the conditions of analysis.

Silk proteins, e.g. spider silk proteins, in comparison to commoncellular proteins, show a quite aberrant amino acid composition. Inparticular, silk polypeptides, e.g. spider silk polypeptides, possesslarge quantities of hydrophobic amino acids such as glycine or alanine,but, for example, no (or only very little) tryptophan. Furthermore, silkpolypeptides, e.g. spider silk polypeptides, contain highly repetitiveamino acid sequences or repetitive units, especially in their large coredomain.

Based on DNA analysis it was shown that all silk polypeptides,particularly spider silk polypeptides, are chains of repetitive unitswhich further comprise a limited set of distinct shorter peptide motifs.The expressions “shorter peptide motif” and “consensus sequence” can beused interchangeably. Generally, the silk consensus sequences,particularly the spider silk consensus sequences, can be grouped intofour major categories: GPGXX, GGX, A_(x) or (GA)_(n) and spacers. Thesecategories of peptide motifs in silk polypeptides, particularly spidersilk polypeptides, have been assigned structural roles. For example, ithas been suggested that the GPGXX motif is involved in a β-turn spiral,probably providing elasticity. The GGX motif is known to be responsiblefor a glycine-rich 3₁-helix. Both GPGXX and GGX motifs are thought to beinvolved in the formation of an amorphous matrix that connectscrystalline regions, thereby providing elasticity of the fiber.Alanine-rich motifs typically contain 6-9 residues and have been foundto form crystalline β-sheets. The spacers typically contain chargedgroups and separate the iterated peptide motifs into clusters.

A fifth category is represented by a non-repetitive (NR) region at theamino- and carboxyl termini of the proteins, often representing chainsof about 100 amino acids. It is thought that the NR carboxy-terminimight play a crucial role during assembly of the silk fiber.

The term “silk particles”, e.g. “spider silk particles”, as used hereinrefers to micro- or submicro-sized spherical structures which are formedby protein aggregation under certain conditions. Preferably, the silkparticles, e.g. spider silk particles, have a smooth surface, aremechanical stable and/or are not water soluble. It is also preferredthat the silk particles, e.g. spider silk particles, have a homogenousmatrix, preferably without any clearly visible inclusions (e.g.determined via electron microscopy). In this respect, it should be notedthat said inclusions may be air and polypeptides which are not relatedto silk polypeptides. In this respect, it should be noted that saidinclusions do not encompass the at least one compound which is loadedinto and/or onto the silk particles according to the present invention.

The silk particles, e.g. spider silk particles, according to theinvention comprise one or more silk polypeptides, e.g. spider silkpolypeptides, each comprising at least two identical repetitive units.

As used herein, the term “one or more silk polypeptides”, e.g. “one ormore spider silk polypeptides”, preferably means that the silk particle,e.g. spider silk particle, does not additionally contain any otherrepetitive proteins, such as elastines, which do not relate, forexample, to spider silk.

The silk polypeptide according to the invention may be any silkpolypeptide known to one skilled in the art. The silk polypeptide,according to the invention may, for example, be any naturally occurringwild type polypeptide sequence, e.g. the polypeptide sequence of anarthropod silk polypeptide, such as a spider silk polypeptide or aninsect silk polypeptide, or a mussel silk polypeptide.

The silk polypeptide, e.g. the spider silk polypeptide, according to theinvention may also be a synthetic or recombinant silk polypeptide, e.g.a synthetic or recombinant spider silk polypeptide, which sequence maybe derived from one or more authentic silk protein sequences, e.g.spider silk protein sequences.

Preferably, the silk polypeptide comprises a sequence derived from anarthropod silk polypeptide, such as a spider silk polypeptide or aninsect silk polypeptide. The silk polypeptide may also comprise asequence derived from a mussel silk polypeptide.

It is preferred that the spider silk polypeptide comprises a sequencederived from a major ampullate gland polypeptide (MaSp), such as adragline spider silk polypeptide, a minor ampullate gland polypeptide(MiSp), a flagelliform polypeptide, an aggregate spider silkpolypeptide, a tubuliform spider silk polypeptide, an aciniform spidersilk polypeptide or a pyriform spider silk polypeptide.

It is further preferred that the insect silk polypeptide comprises asequence derived from a silk polypeptide of Lepidoptera. Morepreferably, the insect silk polypeptide comprises a sequence derivedfrom a silk polypeptide of Bombycidae, most preferably of Bombyx mori.

Useful spider silk polypeptides in the framework of the presentinvention are describdd in the literature, e.g. in the review article ofR. V. Lewis (2006) Spider Silk: Ancient ideas for new biomaterials,Chem. Rev. 106:3762-3774. The amino acid sequences (and correspondingnucleic acid sequences) of spider silk polypeptides which can be used inthe present invention can also be found in the databases known to theskilled person, e.g. the NCBI database. Some examples of such spidersilk polypeptide sequences are given below in the sequence listing inSEQ ID NOs. 49 to 96. In detail, SEQ ID NOs: 49 to 52 represent spidersilk polypeptide sequences of araneus diadematus fibroin 1 to 4, SEQ IDNOs: 53 to 64 represent spider silk polypeptide sequences of majorampullate spidroin I (MaSp I), SEQ ID NOs: 65 to 78 represent spidersilk polypeptide sequences of major ampullate spidroin II (MaSp II), SEQID NOs: 79 to 81 represent sequences of minor ampullate silkpolypeptides, SEQ ID NOs: 82 to 89 represent sequences of flagelliformsilk polypeptides, SEQ ID NO: 90 represents the spider silk polypeptidesequence of aciniform spidroin, SEQ ID NO: 91 to 96 represent the spidersilk polypeptide sequences of tubuliform spidroin.

It is particularly preferred that the spider silk polypeptide sequencesare derived from spider silk dragline (major ampullate), flagelliform,piriform, tubuliform, minor ampullate, aggregate silk, or aciniformproteins. The spider silk sequences may be derived from orb-web spidersuch as Araneidae and Araneoids. More preferably, the spider silksequence can be derived from the group consisting of the followingspiders:

Arachnura higginsi, Araneus circulissparsus, Araneus diadematus, Argiopepicta, Banded Garden Spider (Argiope trifasciata), Batik Golden WebSpider (Nephila antipodiana), Beccari's Tent Spider (Cyrtophorabeccarii), Bird-dropping Spider (Celaenia excavata), Black-and-WhiteSpiny Spider (Gasteracantha kuhlii), Black-and-yellow Garden Spider(Argiope aurantia), Bolas Spider (Ordgarius furcatus), BolasSpiders—Magnificent Spider (Ordgarius magnificus), Brown Sailor Spider(Neoscona nautica), Brown-Legged Spider (Neoscona rufofemorata), CappedBlack-Headed Spider (Zygiella calyptrata), Common Garden Spider(Parawixia dehaani), Common Orb Weaver (Neoscona oxancensis), Crab-likeSpiny Orb Weaver (Gasteracantha cancriformis (elipsoides)), Curved SpinySpider (Gasteracantha arcuata), Cyrtophora moluccensis, Cyrtophoraparnasia, Dolophones conifera, Dolophones turrigera, Doria's SpinySpider (Gasteracantha doriae), Double-Spotted Spiny Spider(Gasteracantha mammosa), Double-Tailed Tent Spider (Cyrtophoraexanthematica), Aculeperia ceropegia, Eriophora pustuloses; FlatAnepsion (Anepsion depressium), Four-spined Jewel Spider (Gasteracanthaquadrispinosa), Garden Orb Web Spider (Eriophora transmarina), GiantLichen Orbweaver (Araneus bicentenarius), Golden Web Spider (Nephilamaculata), Hasselt's Spiny Spider (Gasteracantha hasseltii), Tegenariaatrica, Heurodes turrita, Island Cyclosa Spider (Cyclosa insulana),Jewel or Spiny Spider (Astracantha minax), Kidney Garden Spider (Araneusmitificus), Laglaise's Garden Spider (Eriovixia laglaisei), Long-BelliedCyclosa Spider (Cyclosa bifida), Malabar Spider (Nephilengysmalabarensis), Multi-Coloured St Andrew's Cross Spider (Argiopeversicolor), Ornamental Tree-Trunk Spider (Herennia ornatissima), OvalSt. Andrew's Cross Spider (Argiope aemula), Red Tent Spider (Cyrtophoraunicolor), Russian Tent Spider (Cyrtophora hirta), Saint Andrew's CrossSpider (Argiope keyserlingi), Scarlet Acusilas (Acusilas coccineus),Silver Argiope (Argiope argentata), Spinybacked Orbweaver (Gasteracanthacancriformis), Spotted Orbweaver (Neoscona domiciliorum), St. AndrewsCross (Argiope aetheria), St. Andrew's Cross Spider (ArgiopeKeyserlingi), Tree-Stump Spider (Pols illepidus), Triangular Spider(Arkys clavatus), Triangular Spider (Arkys lancearius), Two-spinedSpider (Poecilopachys australasia), Nephila species, e.g. Nephilaclavipes, Nephila senegalensis, and Nephila madagascariensis. The spidersilk sequence may also be derived from widow spiders such as brown widowspiders (Latrodectus geometricus), black widow spiders or grey widowspiders.

As used herein “a recombinant silk polypeptide”, e.g. “a recombinantspider silk polypeptide”, may comprise

-   -   a) one or more synthetic repetitive silk protein, e.g. spider        silk protein, sequences and/or    -   b) one or more authentic non-repetitive silk protein, e.g.        spider silk protein, sequences.

It is also clear that a recombinant silk polypeptide, e.g. spider silkpolypeptide, may comprise sequences derived from different species, e.g.spider species. For example, the synthetic repetitive silk proteinsequences may be derived from one species, while the one or morenon-repetitive silk protein sequences, e.g. spider silk proteinsequences, may be derived from another species. It is also possible todesign a recombinant silk polypeptide, e.g. spider silk polypeptide,comprising one or more repetitive sequences which are derived fromdifferent species, e.g. spider species.

The term “synthetic repetitive sequence” as used herein is to beunderstood as a recombinant protein sequence which is not a natural silkprotein sequence, e.g. spider silk protein sequence, but maynevertheless be derived from the repetitive units comprising consensussequences or motifs of authentic silk proteins, e.g. spider silkproteins. The recombinant silk polypeptide, e.g. spider silkpolypeptide, according to the present invention comprises at least twoidentical repetitive units. A repetitive unit may further compriseeither one or more monomeric sequence modules or one or more shortpeptide motifs.

A system for producing recombinant spider silk proteins has already beendeveloped and described in WO 2007/025719. In this expressions system,single building blocks, so called modules, can be freely combined toyield synthetic spider silk polypeptides. Modules of this type are alsodescribed in Hümmerich et al. [Hümmerich, D. (2004): “Primary structureelements of dragline silks and their contribution to protein solubilityand assembly,” Biochemistry 43, 13604-13612.] Spider silk monomericsequence modules are further described in WO 2007/025719 in detail.Suitable vectors and plasmids for the expression of silk polypeptide,e.g. spider silk polypeptide, sequences in a host cell are described inthese references.

In brief, the recombinant silk proteins, preferably spider silkproteins, can be produced in a host by expression of suitable nucleicacids or vectors. The host may be for example a prokaryotic cell.Preferred prokaryotic organisms are E. coli or Bacillus subtilis.

The host may also be a eukaryotic cell. Preferred eukaryotic cells aremammalian cells, plant cells, yeast cells or insect cells. Preferredmammalian cells are, for instance, CHO, COS, HeLa, 293T, HEH or BHKcells. If yeast cells are used, preferred organisms are Saccharomycescerevisiae, Schizosaccaromyces pombe, Pichia pastoris, Candida albicansor Hansenula polymorpha. Preferred insect cells are cells fromLepidoptera insects, more preferably cells from Spodoptera frugiperdaand from Trichoplusia ni. Most preferably, the insect cell is a Sf0,Sf21 or high five cell. If the host is a plant cell, the cell ispreferably derived from tobacco, potato, corn and tomato.

Preferably, the basis of the sequence modules are the genes ADF-3 andADF-4 of the spider Araneus diadematus as well as the gene FLAG of thespider Nephila clavipes. The genes ADF-3 and ADF-4 encode for proteinswhich form the dragline thread of the spider. Both proteins, ADF-3 andADF-4 belong to the class of MaSp II proteins (major ampullate spidroinII). The gene FLAG encodes for a flagelliform silk protein.

Modules suitable for the assembly of a synthetic silk protein construct,e.g. spider silk protein construct, are for example:

Modul A: (SEQ ID NO: 20) GPYGPGASAA AAAAGGYGPG SGQQ, Modul C:(SEQ ID NO: 21) GSSAAAAAAA ASGPGGYGPE NQGPSGPGGY GPGGP, Modul Q:(SEQ ID NO: 22) GPGQQGPGQQ GPGQQGPGQQ, Modul K: (SEQ ID NO. 23)GPGGAGGPYGPGGAGGPYGPGGAGGPY, Modul sp: (SEQ ID NO: 24)GGTTIIEDLD ITIDGADGPITISEELTI, Modul X: (SEQ ID NO: 27)GGAGGAGGAG GSGGAGGS, and Modul Y: (SEQ ID NO: 28)GPGGAGPGGY GPGGSGPGGY GPGGSGPGGY.

Further suitable modules are for example:

Modul A^(C): (SEQ ID NO: 29) GPYGPGASAA AAAAGGYGPG CGQQ, Modul A^(K):(SEQ ID NO: 30) GPYGPGASAA AAAAGGYGPG KGQQ, Modul CC: (SEQ ID NO: 31)GSSAAAAAAA ASGPGGYGPE NQGPCGPGGY GPGGP, Modul C^(K1): (SEQ ID NO: 32)GSSAAAAAAA ASGPGGYGPE NQGPKGPGG Y GPGGP, Modul C^(K2): (SEQ ID NO: 33)GSSAAAAAAA ASGPGGYGPK NQGPSGPGGY GPGGP, and Modul C^(KC):(SEQ ID NO: 34) GSSAAAAAAA ASGPGGYGPK NQGPCGPGGY GPGGP.

Said modules may further comprise the following amino terminal and/or acarboxy terminal TAGs:

TAG^(CYS1): (SEQ ID NO: 35) GCGGGGGGSGGGG, TAG^(CYS2): (SEQ ID NO: 36)GCGGGGGG, TAG^(CYS3): (SEQ ID NO: 37) GCGGSGGGGSGGGG, TAG^(LYS1):(SEQ ID NO: 38) GKGGGGGGSGGGG, and TAG^(LYS2): (SEQ ID NO: 39) GKGGGGGG.

Still further modules which can be present in the silk polypeptides,e.g. spider silk polypeptides, of the invention are described in theprior art literature. In this context, it is referred to internationalpatent application WO 2008/155304 A1 and herein in particular to SEQ IDNO: 2 (R16) and SEQ ID NO: 4 (S16) in the sequence listing of WO2008/155304 A1.

The amino acid sequences of the above modules and TAGs comprise one ormore lysine and/or cysteine residues. The modules and/or TAGs are,therefore, capable of producing modified silk proteins, particularlyspider silk proteins. Modified silk proteins, particularly spider silkpolypeptides, are described in detail in WO 2007/025719. It has to beunderstood that compounds may also be coupled to the modified silkproteins, particularly spider silk proteins, via their lysine and/orcysteine residues.

Further, the above described modules can be freely combined in order toyield a suitable silk polypeptide, e.g. spider silk polypeptide,according to the invention. The number of modules of a silk polypeptide,e.g. spider silk polypeptide, is generally not restricted. Preferably,the recombinant silk polypeptides, e.g. spider silk polypeptides, maycomprise between 2 and 50 modules, more preferably between 10 and 40,and most preferably between 15 and 35 modules.

For example, a synthetic repetitive sequence may comprise at least twoof the combinations (AQ) and/or (QAQ) as repetitive units.

If the synthetic repetitive sequence is derived from ADF-4, it maycomprise at least two identical repetitive units, each comprising theamino acid sequence of module C (SEQ ID NO: 21) or a variant thereof.For example, the resulting sequence may be C₁₆ or C₃₂, i.e. comprising16 or 32 repetitive units, respectively. In this respect it should benoted that the terms “eADF4(C16)” and “C₁₆” are interchangeable be usedin the context of the present invention and have the same meaning.

A compound which is well-suited for efficient loading of the silkparticles, e.g. spider silk particles, is preferably sufficiently smallin size. In a preferred embodiment of the invention, the compound has amolecular weight of 50 Da or about 50 Da to 20 kDa or about 20 kDa; or50 Da or about 50 Da to 10 kDa or about 10 kDa, preferably 50 Da orabout 50 Da to 6 kDa or about 6 kDa, more preferably 50 Da or about 50Da to 4 kDa or about 4 kDa and most preferably 50 Da or about 50 Da to 1kDa or about 1 kDa, e.g. 50 Da, 100 Da, 150 Da, 200 Da, 250 Da, 300 Da,350 Da, 400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650 Da, 700 Da, 750 Da,800 Da, 850 Da, 900 Da, 950 Da, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa,3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa,8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa,15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, or 20 kDa.

Further, a compound which is well-suited for efficient loading of thesilk particles, e.g. spider silk particles, is preferably water-soluble.

Furthermore, a preferred compound according to the invention may be anycompound, which is a small and water-soluble compound, preferably havinga molecular weight of between about 50 Da and 20 kDa, more preferably 50Da to 10 kDa or 50 Da to 6 kDa and most preferably 50 Da to 4 kDa or 50Da to 1 kDa (see above).

The term “soluble” as used herein means that a solid, liquid or gaseouschemical substance called solute is able to dissolve in a liquid solventto form a homogeneous solution. Generally, the solubility of a substancestrongly depends on the solvent that is used as well as on temperatureand pressure. The extent of the solubility of a substance in a specificsolvent is measured as the saturation concentration where adding moresolute does not increase the concentration of the solution.

A “solvent” is a liquid which can dissolve gases, other liquids or solidmaterials without chemical reactions between dissolved matter anddissolving liquid taking place.

A “water-soluble” compound is usually any ionic compound (or salt) whichis able to dissolve in water. Generally, the underlying solvation arisesbecause of the attraction between positive and negative charges of thecompound with the partially-negative and partially positive charges ofthe H₂O-molecules, respectively. Substances or compounds which dissolvein water are also termed “hydrophilic” (“water-loving”). Watersolubility (S_(W)), also known as aqueous solubility, is the maximumamount of a substance that can dissolve in water at equilibrium at agiven temperature and pressure. Generally, the limited amount is givenby the solubility product.

Following the definition of solubility in the European Pharmacopoeia,“sparingly solube” means that the approximate volume of solvent inmillilitres per gram of solute is from 30 to 1.00 (at a temperaturebetween 15° C. and 25° C.); “soluble” means that the approximate volumeof solvent in millilitres per gram of solute is from 10 to 30 (at atemperature between 15° C. and 25° C.), “freely soluble” means that theapproximate volume of solvent in millilitres per gram of solute is from1 to 10 (at a temperature between 15° C. and 25° C.), and “very soluble”means that the approximate volume of solvent in millilitres per gram ofsolute is less than 1 (at a temperature between 15° C. and 25° C.).

Accordingly, in the context of the present invention “water-soluble”means a water solubility of 10 g compound or more per 1 liter of water.Preferably, the water solubility is at least 20 g, at least 30 g, atleast 40 g, and at least 50 g compound per 1 liter of water, morepreferably at least 60 g, at least 70 g, at least 80 g, at least 90 andat least 100 g compound per 1 liter of water, and most preferably atleast 200 g, at least 300 g, at least 400 g, at least 500 g, and atleast 800 g compound per 1 liter of water. Very water-soluble compoundsthat can be used in the present invention even have a water solubilityof 1 g/ml or more.

Compounds which are water soluble typically comprise the followingchemical groups: cationic groups such as metallic cations, ammoniumcations and/or anionic groups such as acetate, nitrate, chloride,bromide, iodide or sulphate.

Typical measures for water solubility used in organic chemistry and thepharmaceutical sciences are a partition—(P) or distribution coefficient(D), which give the ratio of concentrations of a compound in the twophases of a mixture of two immiscible solvents at equilibrium.

For example, the octanol-water partition coefficient (log P _(o/w)) istypically used to estimate the water solubility of substances and isdefined as the ratio of a compound's concentration in the octanol phaseto its concentration in the aqueous phase of a two-phase octanol/watersystem. Thus, the octanol-water partition coefficient provides a measureof the lipophilic versus hydrophilic nature of a compound. In general,log P tends to be large for compounds with extended non-polar-structuresand small for a compound of a hydrophilic nature. Methods fordetermining the log P value of a compound are for example the shakeflask (or tube) method, HPLC or electrochemical methods such as ITIES(Interfaces between two immiscible electrolyte solutions).

There are many log P calculators or predictors available bothcommercially and for free on the internet, e.g. Chemistry DevelopmentKit, JOELib, ACD/LogP-DB, ACD/Log P Freeware, Simulations Plus—S+logP,ALOGPS, Molecular Property Explorer, Free online log P calculationsusing ChemAxon's Marvin and Calculator Plugins, miLogP free log P,PreADMET, XLOGP3.

Preferably, the log P value can be predicted using ACDlogP-Software(available at Advanced Chemistry Development, ACD/labs,http://www.acdlabs.com).

In the context of the present invention, the compound preferably has anoverall hydrophilic nature. Compounds suitable for loading of the silkparticles, preferably spider silk particles, comprise also amphiphilicsubstances such as proteins or peptides. According to preferredembodiments, the log P value of the compound is less than 6, preferablyless than 5.5, even more preferably less than 5, and most preferablyless than 4.5, e.g. less than 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1or 0.5.

Further, the distribution between a hydrophobic and a hydrophilic phaseof two different species of a specific compound, i.e. the native and theprotonated form, can be described by its apparent distributioncoefficient (log D), which can be calculated using the followingequations:for acids: log D=log P−log(1+10^(pH-pKa)), andfor bases: log D=log P−log(1+10^(pKa-pH)).

In the context of the present invention, compounds are preferred whichpossess a distribution coefficient (log D) of more than −2, preferablyof more than −1.5, more preferably of more than −1, even more preferablyof more than −0.5 and most preferably of more than 0.

In preferred embodiments of the invention, the silk particles,preferably spider silk particles, provided in step i) are produced bythe steps of

-   -   a) providing an aqueous solution comprising one or more silk        polypeptides, preferably spider silk polypeptides, comprising at        least two identical repetitive units,    -   b) triggering aggregation of the silk polypeptides, preferably        spider silk polypeptides, to form silk particles, preferably        spider silk particles, and    -   c) separating the silk particles, preferably spider silk        particles, by phase separation.

Generally, starting from an aqueous solution, aggregation of the silkpolypeptides, preferably spider silk polypeptides, can be triggeredunder certain conditions to form silk particles, preferably spider silkparticles.

“Aggregation” or “phase separation” as used herein means the particleformation due to a salting-out mechanism which in particular can beconsidered as a liquid-liquid phase separation. The “one-phase state” isthe initial state displayed by a solution of monomeric and intrinsicallyunfolded protein molecules. For example, changing constraints such asthe ionic strength by addition of kosmotropic ions alters the freeenergy of the system and leads to phase separation into protein-rich andsolvent-rich phases. This phase-separated state is energeticallyfavoured and the protein concentration in the “protein phase” increasesto a critical level. Upon reaching the critical concentration fornucleation, several structured nuclei are formed simultaneously in theprotein-rich phase. The nuclei start to grow in a spherical manner,interacting with additional monomers and thereby converting theirstructure. Spherical growth stops when the protein concentration in theprotein-rich phase is below the equilibrium of solubility. Hence thesphere size does not increase further. Phase separation thus means thatprotein-rich and solvent-rich phases are separated. Without being boundto a theory, the sphere size is generally dependent on proteinconcentration and mixing conditions. There exist however various othermethods in the art for triggering aggregation of proteins.

The process of microsphere assembly is typically monitored bylight-scattering after initiation of aggregation. In particular, thecolloidal stability of the resulting particles can be analysed bymeasuring the intensity of scattered light, at a certain wavelength.Also the mean particle size and particle size distribution can bedetermined by laser diffraction, also called static light scattering(SLS). Generally, laser diffraction utilizes the theories of Mie andFraunhofer to determine particle size distribution from a lightscattering pattern. These depend upon analysis of the “halo” ofdiffracted light produced when a laser beam passes through a dispersionof particles in air or in a liquid. The angle of diffraction increasesas particle size decreases. The mass and the root mean square radius ora measure of geometric size can be determined using this technique on amega Dalton scale. Thus, according to the Mie theory, the intensity ofscattered light in forward direction increases with increasing particlesize. The onset of aggregation in dilute dispersions can thus bedetected by intensity of scattered light in forward direction.

The obtained silk particles, preferably spider silk particles, may alsobe analysed using methods such as scanning electron microscopy (SEM) andFourier transform infrared spectroscopy (FTIR). A further description ofthese methods can be found in the description and in the examples below.

In order to analyze the morphology and structure of the silk particles,preferably spider silk particles, scanning electron microscopy (SEM) cantypically be employed. The scanning electron microscope (SEM) is a typeof electron microscope that images the sample surface by scanning itwith a high-energy beam of electrons in a raster scan pattern. Theelectrons interact with the atoms that make up the sample producingsignals that contain information about the sample's surface topography,composition and other properties such as electrical conductivity.

Further characteristics such as the secondary structure of the obtainedsilk particles, preferably spider silk particles, can for example beanalyzed by Fourier transform infrared spectroscopy (FTIR). Thetechnique is based on the fact that bonds and groups of bonds vibrate atcharacteristic frequencies. A molecule that is exposed to infrared raysabsorbs infrared energy at frequencies which are characteristic for thatmolecule. During FTIR analysis, a spot on the specimen is subjected to amodulated IR beam. The specimen's transmittance and reflectance of theinfrared rays at different frequencies is translated into an IRabsorption plot consisting of reverse peaks. The resulting FTIR spectralpattern is then analyzed and matched with known signatures of identifiedmaterials in the FTIR library. For example, peaks at 1648-1660 cm⁻¹,1625-1640 cm⁻¹ and 1660-1668 cm⁻¹, can be assigned to α-helical,β-sheets and β-turn structures of the silk polypeptides, e.g. spidersilk polypeptides, respectively.

After phase separation, the produced particles can be separated byroutine methods such as centrifugation. The prepared particles maysubsequently be washed and incubated with a compound of interest. Aswill be mentioned below, the particles may also be stored, for example,in a dried or lyophilized form.

The particles according to the invention usually consist of a smoothsurface and a solid matrix. In the context of the invention, the term“surface” defines the outer sphere of the particle, which includes thosesphere sections that are directly exposed to the surrounding space, i.e.the surrounding medium. Although the particles appear rather smooth anduniform, their surfaces on the sub-microscopic level reveal a thinmantle with irregular and diffuse structures. A surface, thus,delineates the outermost layer of the particle which shares an interfacewith the surrounding medium and at which adhesion and bidirectionaldiffusion of the compound molecules may occur.

The term “matrix” as used herein defines the inner sphere of the silkparticle, preferably spider silk particle, which is not the surface,i.e. which according to the above definition does not include anyinterface to the surrounding medium. The matrix is to be understood as asolid sphere having a radius and accordingly a volume usually smallerthan that of the particle.

The volume of the matrix is usually more than 50% of the total volume,preferably more than 60%, 70%, 80%, 90%, most preferably more than 95%,e.g. more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%.

The term “loading” in the context of the present invention means thenon-covalent binding of a compound to a silk particle, preferably spidersilk particle, via adhesion to the surface and via diffusion and/orpermeation and subsequent adhesion to the matrix of the silk particle,preferably spider silk particle, wherein preferably at least 40%, morepreferably at least 50%, 60%, 70%, 80%, 90%, or 95% of the loadedcompound is located within the matrix of the silk particle, preferablyspider silk particle. The non-covalent binding mentioned herein iscaused, for example, by electrostatic interactions, hydrophilicinteractions (hydrogen-bonds) and/or hydrophobic interactions (van derWaals forces).

There are several ways to determine the “loading” of a silk particle,e.g. spider silk particle. For example, one way is to determine theresidual concentration of the compound in the supernatant after a periodof time of incubation with the silk particles, e.g. spider silkparticles. As will be explained in more detail in the examples below,the residual concentrations of a compound may be measured using UV-Visspectroscopy.

To determine the percentage of the loaded compound which is adhered tothe matrix of the silk particle, preferably spider silk particle, thefollowing model calculation can be used: For calculation of thetheoretical maximal adhesion capacity of a silk particle, theclosest/densest sphere packing of a compound on the silk particle istaken. By means of the medians of the silk particle and the compound,the surface of the silk particle and the compound can be determined.Corresponding to the surface, the maximum amount of compound which canbe in direct contact to the particle surface can be determined. Amonolayer of compound will be assumed as closest/densest sphere packingof a compound on the silk particle. More than one layer of compoundloaded to the surface of a silk particle is unlikely, due to theelectrostatic repulsion between, for example, two positively chargedcompounds. On the basis of ratio of totally loaded compound to compoundloaded to the surface of a silk particle, the percentage of loadedcompound into the matrix can be determined.

The non-covalent binding of a positively charged compound to the surfaceof a negatively charged particle via adhesion decreases the absolutevalue of zeta-potential in contrast to the non-covalent binding of apositively charged compound to the matrix of a negatively chargedparticle via diffusion and/or permeation and subsequent adhesion, whichdoes not substantially alter the zeta-potential of the particle.

The high percentage of a particle-bound (adhered) compound compared to afree compound in solution results in a very high loading efficiency.

The adhered compound is protected in the matrix of the silk particle andcan, therefore, safely be stored for several weeks for later use.

The adhered compound can also be efficiently and constantly releasedfrom the silk particle without the requirement of degradation of thesilk particle—in contrast to an irreversible/sterically-trapped boundcompound. The compound can be steadily released over a time period ofdays to weeks—in contrast to the burst release of compounds which areexclusively adhered at the surface of the silk particle, preferablyspider silk particle.

In further preferred embodiments, the compound is able to permeate intothe matrix of the silk particles, preferably spider silk particles. Theterm “permeate” in physics and engineering generally means thepenetration of a permeate, which can be a liquid, gas or vapour, througha solid and is dependent on the material's intrinsic permeability. Inparticular, permeation of a compound according to the present inventionoccurs by molecular diffusion, which by definition is a net transport ofmolecules from a region of higher concentration to one of lowerconcentration by random molecular motion. It has to be understood thatduring the process of permeation the compound at first adheres to thesurface of the silk particles, preferably spider silk particles, andthen permeates the surface layer into the matrix of the silk particles,preferably spider silk particles, by molecular diffusion.

As used herein the term “is able to permeate into the silk particles”,e.g. “is able to permeate into the spider silk particles” thus meansthat the compound is able to soak into the silk matrix, e.g. spider silkmatrix, by molecular diffusion. Whether a compound is able to permeateinto the matrix of the silk particles, e.g. spider silk particles, canbe determined using several methods.

For example, one way is to determine the residual concentration of thecompound in the supernatant after a period of time of incubation withthe silk particles, e.g. spider silk particles. As will be explained inmore detail in the examples below, the residual concentrations of acompound may be measured using UV-Vis spectroscopy.

Generally, Ultraviolet-visible spectroscopy or ultraviolet-visiblespectrophotometry (UV-Vis or UV/Vis) involves the spectroscopy ofphotons in the UV-visible region. This technique thus uses light in thevisible and adjacent (near ultraviolet (UV) and near infrared (NIR))ranges. In this region of the electromagnetic spectrum, molecules in themeasured sample undergo electronic transition. The UV-Vis-spectroscopyis, therefore, generally used in the quantitative determination ofsolution of organic compounds. Within the context of the presentinvention, the encapsulation efficiency and loading of the silkparticles, preferably spider silk particles, can be determined usingUV-Vis spectroscopy and calculated on basis of the following equations:

${{encapsulation}\mspace{14mu}{efficiency}\mspace{14mu}\left( {w\text{/}w\mspace{14mu}\%} \right)} = {\frac{{amount}\mspace{14mu}{of}\mspace{14mu}{compound}\mspace{14mu}{in}\mspace{14mu}{particles}}{{compound}\mspace{14mu}{initially}\mspace{14mu}{added}} \times 100}$

For example, the “encapsulation efficiency” is calculated to be 66% withthe following data: amount of compound non-covalently bound to thesurface and to the matrix of the silk particle: 0.1 g, amount ofcompound initially added: 0.15 g.

${{{encapsulation}\mspace{14mu}{efficiency}} = {\frac{0,{1\mspace{14mu} g}}{0,{15\mspace{14mu} g}}0}},{66 = {66\%}}$${{loading}\mspace{14mu}\left( {w\text{/}w\mspace{14mu}\%} \right)} = {\frac{{amount}\mspace{14mu}{compound}\mspace{14mu}{in}\mspace{14mu}{particles}}{{amount}\mspace{14mu}{of}\mspace{14mu}{particles}} \times 100}$

For example the “loading” is calculated to be 10% with the followingdata: amount of compound non-covalently bound to the surface and to thematrix of the silk particle: 0.1 g, amount of compound initially added:1.0 g.

${{loading} = {\frac{0,{1\mspace{14mu} g}}{1,{0\mspace{14mu} g}} = 0}},{1 = {10\%}}$

The terms “encapsulation efficiency” and “loading efficiency” are usedinterchangeable in the context of the present invention and have,therefore, the same meaning.

In a preferred embodiment, at least 10%, 20%, or 30%, more preferably atleast 40%, 50%, or 60%, and most preferably at least 70%, 80%, 90%, or95% of the compound is loaded to the silk particles (to the silk surfaceand to the matrix), preferably spider silk particles, e.g. at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%.

In another preferred embodiment, at least 40%, preferably at least 50%,60%, 70%, 80%, 90%, or 95% of the loaded compound is located within thematrix of the silk particles, preferably spider silk particles, e.g. atleast 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, or 95%.

In a more preferred embodiment, at least 10%, 20%, or 30%, morepreferably at least 40%, 50%, or 60%, and most preferably at least 70%,80%, 90%, or 95% of the compound is loaded to the silk particles (to thesilk surface and to the matrix), preferably spider silk particles, e.g.at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%,wherein at least 40%, preferably at least 50%, 60%, 70%, 80%, 90%, or95% of the loaded compound is located within the matrix of the silkparticles, preferably spider silk particles, e.g. at least 40, 41, 42,43, 44, 45, 46; 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%.

One further criterion which gives an indication whether a compound isable to permeate into the interior of the silk particles, e.g. spidersilk particles, is the zeta-potential. As will be apparent from theexamples, monitoring of the zeta-potential of the particles mayparticularly indicate whether a compound is merely bound at the particlesurface or is able to diffuse into the interior. Zeta-potentialmeasurements are especially applicable when the produced silk particles,e.g. spider silk particles, possess an overall net charge.

As used herein, “zeta potential” or “ζ-potential” is the electricalpotential in the interfacial double layer (DL) at the location of theslipping plane versus a point in the bulk fluid away from the interface.In other words, zeta potential is the potential difference between thedispersion and the stationary layer of fluid attached to a dispersedparticle.

In particular, the permeation process can be monitored on the basis ofthe observed changes of the zeta potential during loading. A change ofthe zeta potential is thus a measure for the permeation of a compoundinto the matrix of the silk particles, e.g. spider silk particles.

The mechanism of permeation according to the present invention is, thus,clearly distinguishable from mechanisms such as encapsulation ofcompounds as has been described in the art. For instance, theencapsulation as described in patent applications WO 2007/082936 and WO2007/082923 are is in both cases based on the inclusion of poorlywater-insoluble compounds. However, diffusion of the compound moleculesinto the interior of the particles was not described at all in theseprior art references. Rather, the compounds are enveloped by the spidersilk material during particle formation. For this reason, particleformation and loading of a compound according to the prior art must tobe carried out in one single step.

In contrast, the method of the present invention allows that theincubation step, i.e. loading of a compound, can be carried out during,but preferably also after the step of preparing the silk particles, e.g.spider silk particles. However, this does not mean that these two stepsmust also be carried out consecutively in one continuous process. Asmentioned above, one major advantage of the present invention is thatthe steps of producing and loading of the particles can be carried outseparately, both spatially and at different times.

As mentioned above, the produced silk particles, e.g. spider silkparticles, may be provided separately in a dry form, e.g. in the form ofa powder. Suitable methods such as lyophilisation are known in the art.Lyophilisation may however also occur after the particles were loadedwith a compound. Before use, the dried silk particles, e.g. spider silkparticle's, have to be redispersed, i.e. hydrated with an aqueous liquidor suitable buffer.

Further, the silk particles, e.g. spider silk particles, produced by themethod of the invention generally may have a median size ranging fromseveral nanometers to several hundred micrometers. As mentioned above,particle size and size distribution can be determined using laserdiffraction spectroscopy.

According to preferred embodiments, the silk particles, preferablyspider silk particles, have a median size of between 0.1 μm and 500 μm,preferably of between 0.1 μm and 100 μm, more preferably of between 0.2μm and 20 μm, even more preferably of between 0.2 μm and 1 μm and mostpreferably of between 0.25 μm and 0.7 μm, e.g. 0.1, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,or 500 μm.

As mentioned above, the silk polypeptide, preferably spider silkpolypeptide, according to the invention can be either any naturallyoccurring wild type polypeptide sequence or any synthetic or recombinantsilk polypeptide, preferably spider silk polypeptide, or also a mixturethereof. Preferably, the silk polypeptide, more preferably spider silkpolypeptide, according to the invention is a synthetic or a recombinantsilk polypeptide, more preferably spider silk polypeptide.

A “silk polypeptide”, e.g. “spider silk polypeptide”, as used in thecontext of the present invention may refer to a polypeptide with anamino acid sequence which comprises or consists of at least 20%, 30%,40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably at least 95% andmost preferably 100% of multiple copies of one identical repetitive unit(e.g. A₂, Q₆, or C₁₆, wherein the items 2, 6, or 16 represent the numberof repetitive units) or multiple copies of two or more differentrepetitive units (e.g. (AQ)₂₄, or (AQ)₁₂C₁₆).

The terms “repetitive unit” and “repeat unit” are interchangeable beused in the context of the present invention.

In the context of the present invention, a “repetitive unit” may referto a region which corresponds in amino acid sequence to a region thatcomprises or consists of at least one peptide motif (e.g. AAAAAA (SEQ IDNO: 13) or GPGQQ (SEQ ID NO: 4)) that repetitively occurs within anaturally occurring silk polypeptide (e.g. MaSpI, ADF-3, ADF-4, or Flag)(i.e. identical amino acid sequence) or to an amino acid sequencesubstantially similar thereto (i.e. variational amino acid sequence). Inthis regard “substantially similar” may mean a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over the whole length of the respective referencenaturally occurring amino acid sequence. A “repetitive unit” having anamino acid sequence which is “substantially similar” to a correspondingamino acid sequence within a naturally occurring silk polypeptide (i.e.wild-type repetitive unit) is also similar with respect to itsfunctional properties, e.g. the silk particle comprising the silkpolypeptide which comprises the “substantially similar repetitive unit”can still be loaded with a compound. Preferably, the silk particlecomprising the silk polypeptide which comprises the “substantiallysimilar repetitive unit” is capable of being loaded with a compound sothat at least 20%, preferably at least 40%, more preferably at least50%, 60%, 70%, 80%, 90%, or 95% of the loaded compound is located withinthe matrix of the silk particle. The skilled person can readilydetermine the “loading” of a silk particle (e.g. viaUV-Vis-spectroscopy), in particular the percentage of the compound whichis located within the matrix of silk particles (see, for example,experimental section).

A “repetitive unit” having an amino acid sequence which is “identical”to the amino acid sequence of a naturally occurring silk polypeptide,for example, can be a portion of a silk polypeptide corresponding to oneor more peptid motifs of MaSp I (SEQ ID NO: 43) MaSp II (SEQ ID NO: 44),ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2). A “repetitive unit”having an amino acid sequence which is “substantially similar” to theamino acid sequence of a naturally occurring silk polypeptide, forexample, can be a portion of a silk polypeptide corresponding to one ormore peptide motifs of MaSpI (SEQ ID NO: 43) MaSpII (SEQ ID NO: 44),ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2), but having one or moreamino acid substitution at specific amino acid positions.

The “repetitive unit” does not include the non-repetitive hydrophilicamino acid domain generally thought to be present at the carboxylterminus of naturally occurring silk polypeptides.

A “repetitive unit” according to the present invention may further referto an amino acid sequence with a length of 3 to 200 amino acids, or 5 to150 amino acids, preferably with a length of 10 to 100 amino acids, or15 to 80 amino acids and more preferably with a length of 18 to 60, or20 to 40 amino acids. For example, the repetitive unit according to thepresent invention can have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, or 200 amino acids. Most preferably, the repetitiveunit according to the invention consists of 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 18, 20, 24, 27, 28, 30, 34, 35, or 39 amino acids.

The silk polypeptide according to the present invention may consist ofbetween 6 to 1500 amino acids, or between 200 to 1300 amino acids andmost preferably between 250 to 1200 amino acids, or between 500 to 1000amino acids.

The silk polypeptide according to the present invention may comprise orconsist of between 2 to 80 repetitive units, between 3 to 80 repetitiveunits, or between 4 to 60 repetitive units, more preferably between 8 to48 repetitive units, or between 10 to 40 repetitive units and mostpreferably between 16 to 32 repetitive units. For example, the silkpolypeptide according to the present invention can comprise or consistsof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79 or 80 repetitive units. Most preferably, the silkpolypeptide comprises 4, 8, 12, 16, 24, 32 or 48 repetitive units. Asmentioned above, at least two of the repetitive units comprised in thesilk polypeptide according to the present invention are identicalrepetitive units. Thus, the silk polypeptide according to the presentinvention may comprise multiple copies of one identical repetitive unit(e.g. A₂ or C₁₆, wherein the items 2 or 6 represent the number ofrepetitive units) or multiple copies of two or more different repetitiveunits (e.g. (AQ)₂₄ or (QAQ)₈). For example, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 of the80 repetitive units comprised in the silk polypeptide according to thepresent invention may be identical repetitive units.

The term “consensus sequence” as used in the context of the presentinvention refers to an amino acid sequence which contains amino acidswhich frequently occur in a certain position (e.g. “G”) and wherein,other amino acids which are not further determined are replaced by theplace holder “X”.

According to preferred embodiments, the silk polypeptide, preferablyspider silk polypeptide, comprises, essentially consists of, or consistsof at least two identical repetitive units each comprising at least one,preferably one, consensus sequence selected from the group consisting of

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from the group consisting of        A, S, G, Y, P and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from the group consisting of Y, P, R, S,        A, T, N and Q; and    -   iii) A_(x), wherein x is an integer from 5 to 10.

It is also preferred that the silk polypeptide comprises, essentiallyconsists of, or consists of at least two identical repetitive units eachcomprising at least one, preferably one, amino acid sequence selectedfrom the group consisting of: GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG(SEQ ID NO: 19). The GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO:19) (peptide) motifs have been selected from Resilin (WO 08/155304).Resilin is an elastomeric protein found in most arthropods (arthropoda).It is located in specialised regions of the cuticle, providing lowstiffness and high strength (Elvin et al., Nature (473): 999-1002,2005).

Thus, in a preferred embodiment of the present invention, the silkpolypeptide comprises, essentially consists of, or consists ofrepetitive units each comprising at least one (e.g. 1, 2, 3, 4, 5, 6, 7,8, or 9), preferably one, amino acid sequence selected from the groupconsisting of GPGAS (SEQ ID NO: 5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ IDNO: 7), GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO:4), GPGGG (SEQ ID NO: 10), GPGQG (SEQ ID NO: 40), and GPGGS (SEQ ID NO:11). In another preferred embodiment of the present invention, the silkpolypeptide comprises, essentially consists of, or consists ofrepetitive units each comprising at least one (e.g. 1, 2, 3, 4, 5, 8, 7,or 8), preferably one, amino acid sequence selected from the groupconsisting of GGY, GGP, GGA, GGR, GGS, GGT, GGN, and GGQ. In a furtherpreferred embodiment of the present invention, the silk polypeptidecomprises, essentially consists of, or consists of repetitive units eachcomprising at least one (e.g. 1, 2, 3, 4, 5, or 6), preferably one,amino acid sequence selected from the group consisting of AAAAA (SEQ IDNO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQID NO: 15), AAAAAAAAA (SEQ ID NO: 16), and AAAAAAAAAA (SEQ ID NO: 17).

In another preferred embodiment of the invention, the silk polypeptidecomprises, essentially consists of, or consists of repetitive units eachcomprising at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), preferably one,amino acid sequence selected from the group consisting of GPGAS (SEQ IDNO: 5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO:8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO: 10),GPGQG (SEQ ID NO: 40); GPGGS (SEQ ID NO: 11), GGY, GGP, GGA, GGR, GGS,GGT, GGN, GGQ, AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA(SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO: 16),AAAAAAAAAA (SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQID NO: 19).

Most preferably, the silk polypeptide comprises, essentially consistsof, or consists of repetitive units, which comprise or consist of

-   -   (i) GPGAS (SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG        (SEQ ID NO: 6) as amino acid sequence, preferably in this order,    -   (ii) AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ        ID NO: 7), and GPGGP (SEQ ID NO: 8) as amino acid sequence,        preferably in this order,    -   (iii) GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID        NO: 4) and GPGQQ (SEQ ID NO: 4) as amino acid sequence,    -   (iv) GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA        (SEQ ID NO: 9), and GGP as amino acid sequence, preferably in        this order,    -   (v) AAAAAAAA (SEQ ID NO: 15), GPGQG (SEQ ID NO: 40), and GGR as        amino acid sequence, preferably in this order,    -   (vi) AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN,        and GGR as amino acid sequence, preferably in this order,    -   (vii) GGA, GGA, GGA, GGS, GGA, and GGS as amino acid sequence,        preferably in this order, and/or    -   (viii) GPGGA (SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID        NO: 11), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY        (SEQ ID NO: 7) as amino acid sequence, preferably in this order.

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides mentioned above are identical repetitive units.

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of between 2 to 80 repetitive units, between 3 to 80 repetitiveunits, or between 4 to 60 repetitive units, more preferably between 8 to48 repetitive units, or between 10 to 40 repetitive units and mostpreferably between 16 to 32 repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80repetitive units, each comprising at least one, preferably one,consensus sequence selected from the group consisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from A, S, G, Y, P, and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from Y, P, R, S, A, T, N and Q, more        preferably in each case independently selected from Y, P and Q;        and    -   iii) A_(x), wherein x is an integer from 5 to 10.        As mentioned above, at least two of the repetitive units        comprised in the silk polypeptide according to the present        invention are identical repetitive units.

It is also preferred that the silk polypeptide comprises, essentiallyconsists of, or consists of between 2 to 80 repetitive units, between 3to 80 repetitive units, or between 4 to 60 repetitive units, morepreferably between 8 to 48 repetitive units, or between 10 to 40repetitive units and most preferably between 16 to 32 repetitive units,each comprising at least one, preferably one, amino acid sequenceselected from the group consisting of: GGRPSDTYG (SEQ ID NO: 18) andGGRPSSSYG (SEQ ID NO: 19).

Thus, the silk polypeptide preferably comprises, essentially consistsof, or consists of between 2 to 80 repetitive units, between 3 to 80repetitive units, or between 4 to 60 repetitive units, more preferablybetween 8 to 48 repetitive units, or between 10 to 40 repetitive unitsand most preferably between 16 to 32 repetitive units, each comprisingat least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), preferably one, amino acidsequence selected from the group consisting of GPGAS (SEQ ID NO: 5),GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8), GPGGA(SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGQG (SEQ ID NO: 40), GPGGG (SEQID NO: 10), GPGGS (SEQ ID NO: 11), GGY, GGP, GGA, GGR, GGS, GGT, GGN,GGQ, AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO:14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO: 16), AAAAAAAAAA(SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO:19).

Most preferably, the silk polypeptide comprises, essentially consistsof, or consists of

-   -   (i) repetitive units which comprise or consist of GPGAS (SEQ ID        NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG (SEQ ID NO: 6) as        amino acid sequence, preferably in this order,    -   (ii) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ ID NO: 7), and        GPGGP (SEQ ID NO: 8) as amino acid sequence, preferably in this        order,    -   (iii) repetitive units which comprise or consist of GPGQQ (SEQ        ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4) and GPGQQ        (SEQ ID NO: 4) as amino acid sequence,    -   (iv) repetitive units which comprise or consist of GPGGA (SEQ ID        NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9),        and GGP, as amino acid sequence, preferably in this order,    -   (v) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGQG (SEQ ID NO: 40), and GGR as amino acid        sequence, preferably in this order,    -   (vi) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN, and GGR as amino        acid sequence, preferably in this order,    -   (vii) repetitive units which comprise or consist of GGA, GGA,        GGA, GGS, GGA, and GGS as amino acid sequence, preferably in        this order, and/or    -   (viii) repetitive units which comprise or consist of GPGGA (SEQ        ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), GPGGY        (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY (SEQ ID NO: 7)        as amino acid sequence, preferably in this order.

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides mentioned above are identical repetitive units.

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of

-   -   (i) (GPGXX)_(n) (SEQ ID NO: 3) as a repetitive unit, wherein X        is any amino acid, preferably in each case independently        selected from A, S, G, Y, P, and Q and n is 2, 3, 4, 5, 6, 7, 8,        or 9;    -   ii) (GGX)_(n) as a repetitive unit, wherein X is any amino acid,        preferably in each case independently selected from Y, P, R, S,        A, T, N and Q, more preferably in each case independently        selected from Y, P and Q, and n is 2, 3, 4, 5, 6, 7, or 8;        and/or    -   iii) (A_(x))_(n) as a repetitive unit, wherein x is an integer        from 5 to 10 and n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.        As mentioned above, at least two of the repetitive units        comprised in the silk polypeptide according to the present        invention are identical repetitive units.

It is preferred that the repetitive units are independently selectedfrom module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQID NO: 22), module K (SEQ ID NO: 23), module sp (SEQ ID NO: 24), moduleS (SEQ ID NO: 25), module R (SEQ ID NO: 26), module X (SEQ ID NO: 27),or module Y (SEQ ID NO: 28), or variants thereof (i.e. module Avariants, module C variants, module Q variants, Module K variants,module sp variants, module S variants, module R variants, module Xvariants or module Y variants). The modules A (SEQ ID NO: 20) and Q (SEQID NO: 22) are based on the amino acid sequence of ADF-3 of the spiderAraneus diadematus. Module C (SEQ ID NO: 21) is based on the amino acidsequence of ADF-4 of the spider Araneus diadematus. The modules K (SEQID NO: 23), sp (SEQ ID NO: 24), X (SEQ ID NO: 27) and Y (SEQ ID NO: 28)are based on the amino acid sequence of the flagelliform protein FLAG ofthe spider Nephila clavipes (WO 2006/008163). The modules S (SEQ ID NO:25) and R (SEQ ID NO: 26) are based on Resilin (Arthropoda) (WO2008/155304).

Preferably, the silk polypeptide according to the present inventioncomprises, essentially consists of, or consists of between 2 to 80repetitive units, between 3 to 80 repetitive units, or between 4 to 60repetitive units, more preferably between 8 to 48 repetitive units, orbetween 10 to 40 repetitive units and most preferably between 16 to 32repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units, which areindependently selected from module A (SEQ ID NO: 20), module C (SEQ IDNO: 21), module Q (SEQ ID NO: 22), module K (SEQ ID NO: 23), module sp(SEQ ID NO: 24), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26),module X (SEQ ID NO: 27) or module Y (SEQ ID NO: 28), or variantsthereof (i.e. module A variants, module C variants, module Q variants,module K variants, module sp variants, module S variants, module Rvariants, module X variants or module Y variants). It should be notedthat at least two of the repetitive units comprised in the silkpolypeptide according to the present invention are identical repetitiveunits (modules).

Thus, it is preferred that the silk polypeptide according to the presentinvention comprises, essentially consists of, or consists of (i)repetitive unit(s) consisting of module A and/or repetitive unit(s)consisting of module A variants, (ii) repetitive unit(s) consisting ofmodule C and/or repetitive unit(s) consisting of module C variants,(iii) repetitive unit(s) consisting of module Q and/or repetitiveunit(s) consisting of module Q variants, (iv) (a) repetitive unit(s)consisting of module A and repetitive unit(s) consisting of module Q,(b) repetitive unit(s) consisting of module A and repetitive unit(s)consisting of module Q variants, (c) repetitive unit(s) consisting ofmodule A variants and repetitive unit(s) consisting of module Q, (d)repetitive unit(s) consisting of module A variants and repetitiveunit(s) consisting of module Q variants, (v) (a) repetitive units)consisting of module A and repetitive unit(s) consisting of module C,(b) repetitive unit(s) consisting of module A and repetitive unit(s)consisting of module C variants, (c) repetitive unit(s) consisting ofmodule A variants and repetitive unit(s) consisting of module C, (d)repetitive unit(s) consisting of module A variants and repetitiveunit(s) consisting of module C variants, (vi) (a) repetitive unit(s)consisting of module C and repetitive unit(s) consisting of module Q,(b) repetitive unit(s) consisting of module C and repetitive unit(s)consisting of module Q variants, (c) repetitive unit(s) consisting ofmodule C variants and repetitive unit(s) consisting of module Q, (d)repetitive unit(s) consisting of module C variants and repetitiveunit(s) consisting of module Q variants, or (vii) (a) repetitive unit(s)consisting of module A, repetitive unit(s) consisting of module Q andrepetitive unit(s) consisting of module C, (b) repetitive unit(s)consisting of module A, repetitive unit(s) consisting of module Q andrepetitive unit(s) consisting of module C variants, (c) repetitiveunit(s) consisting of module A, repetitive unit(s) consisting of moduleQ variants and repetitive unit(s) consisting of module C, (d) repetitiveunit(s) consisting of module A variants, repetitive unit(s) consistingof module Q and repetitive unit(s) consisting of module C, (e)repetitive unit(s) consisting of module A, repetitive unit(s) consistingof module Q variants and repetitive unit(s) consisting of module Cvariants, (f) repetitive unit(s) consisting of module A variants,repetitive unit(s) consisting of module Q variants and repetitiveunit(s) consisting of module C, (g) repetitive unit(s) consisting ofmodule A variants, repetitive unit(s) consisting of module Q andrepetitive unit(s) consisting of module C variants, (h) repetitiveunit(s) consisting of module A variants, repetitive unit(s) consistingof module Q variants and repetitive unit(s) consisting of module Cvariants.

The modules A, C, Q, K, sp, S, R, X, or Y or variants thereof (i.e.module A variants, module C variants, module Q variants, module Kvariants, module sp variants, module S variants, module R variants,module X variants or module Y variants) can also be combined with eachother in any combination and in any number of each, i.e. module(repetitive unit) A can be combined with module (repetitive unit) Q(i.e. combination AQ), module (repetitive unit) C can be combined withmodule (repetitive unit) Q (i.e. combination CQ), module (repetitiveunit) Q can be combined with module (repetitive unit) A and with module(repetitive unit) Q (i.e. combination QAQ), module (repetitive unit) Acan be combined with module (repetitive unit) A and with module(repetitive unit) Q (i.e. combination AAQ), etc., under the proviso thatthe silk polypeptide used in the method of the present inventioncomprises or consists of at least two repetitive units which areidentical. For example, the silk polypeptide used in the method of thepresent invention can/comprise or consist of A_(n), (AA)_(n), (AQ)_(n),(QA)_(n), Q_(n), (QQ)_(n), (QAQ)_(n), (AQA)_(n), C_(n), (CC)_(n),(CCC)_(n), (CQ)_(n), (QC)_(n), (QCQ)_(n), (CQC)_(n), (AA)_(n)Q_(n),(QQ)_(n)A_(n), (AAA)_(n)Q_(n), (QQQ)_(n)A_(n), (AQQ)_(n), (QQA)_(n),K_(n), sp_(n), S_(n), R_(n), X_(n), Y_(n), (Ksp)_(n), (sPK)_(n),(XY)_(n), (YX)_(n), (XX)_(n), (YY)_(n), (XXX)_(n), (YYY)_(n), (AX)_(n),(XA)_(n), (CX)_(n), (XC)_(n), (QX)_(n), (XQ)_(n), (YQ)_(n), (QY)_(n),(SS)_(n), (SR)_(n), (RS)_(n), or (RR)_(n), wherein n is at least 2,preferably 4, 8, 9, 10, 12, 16, 20, 24, or 32. In case that the silkpolypeptide consists of (AQ)₁₂, it is noted that module (repetitiveunit) A is 12 times present and module (repetitive unit) Q is also 12times present in the silk polypeptide and that, thus, the silkpolypeptide consists of 24 modules (repetitive units). The arrangementof the modules (repeat units) of a silk polypeptide consisting of (AQ)₁₂is as follows: AQAQAQAQAQAQAQAQAQAQAQAQ. Further, in case that the silkpolypeptide of the modules (repeat units) of a silk polypeptide consistsof (QAQ)₈, it is noted that module (repeat unit) A is 8 times presentand module (repetitive unit) Q is 16 times present in the silkpolypeptide and that, thus, the silk polypeptide consists of 24 modules(repetitive units). The arrangement of the modules (repeat units) of asilk polypeptide consisting of (QAQ)₈ is as follows:QAQQAQQAQQAQQAQQAQQAQQAQ.

The silk polypeptide according to the present invention can alsocomprise or consist of (A*Q)_(n), (AQ*)_(n), (A*Q*)_(n), (Q*A)_(n),(QA*)_(n), (Q*A*)_(n), (QAQ*)_(n), (QA*Q)_(n), (Q*AQ)_(n), (QA*Q*)_(n),(Q*A*Q)_(n), (Q*AQ*)_(n), (Q*A*Q*)_(n), (AQA*)_(n), (AQ*A)_(n),(A*QA)_(n), (AQ*A*)_(n), (A*Q*A)_(n), (A*QA*)_(n), (A*Q*A*)_(n), whereinn is at least 2, preferably 4, 8, 9, 10, 12, 16, 20, 24, or 32 andwherein * indicates a module variant, i.e. module A or Q variant.

The terms “combined with each other” or “concatenated with each other”may mean in the context of the present invention that the modules(repetitive units) are directly combined or concatenated with each otheror may mean in the context of the present invention that the modules(repetitive units) are combined or concatenated with each other via oneor more spacer amino acids. In preferred embodiments, the modules(repetitive units) comprised in the silk polypeptide are directlycombined or concatenated with each other. In other preferredembodiments, the modules (repetitive units) comprised in the silkpolypeptide are combined or concatenated with each other via 1 to 25 or1 to 20 spacer amino acids, more preferably via 1 to 15 or 1 to 10spacer amino acids, and most preferably, via 1 to 5 spacer amino acids,i.e. via 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 spacer amino acids. Said spacer aminoacids may be any amino acids naturally occurring in proteins.Preferably, said spacer amino acid is not proline. It is preferred thatthe spacer amino acid(s) contain(s) charged groups. Preferably, thespacer amino acid(s) containing charged groups is (are) independentlyselected from the group consisting of aspartate, glutamate, histidine,and lysine. Said spacer amino acids should be amino acids which do notnegatively affect the ability of a silk particle comprising a silkpolypeptide to receive a compound. The ability of a silk particle toreceive a compound can easily be tested (see above and experimentalsection). Further, said spacer amino acids should be amino acids whichdo not cause steric hindrance, e.g. amino acids having a small size suchas lysine and cysteine.

It is further preferred that the repetitive units are independentlyselected from module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34). The modulesA^(C) (SEQ ID NO: 29), A^(K) (SEQ ID NO: 30), C^(C) (SEQ ID NO: 31),C^(K1) (SEQ ID NO: 32), C^(K2) (SEQ ID NO: 33) and C^(KC) (SEQ ID NO:34) are variants of the module A which is based on the amino acidsequence of ADF-3 of the spider Araneus diadematus and of module C whichis based on the amino acid sequence of ADF-4 of the spider Araneusdiadematus (WO 2007/025719). In module A^(C) (SEQ ID NO: 29) the aminoacid S (serine) at position 21 has been replaced by the amino acid C(cysteine), in module A^(K) (SEQ ID NO: 30) the amino acid S at position21 has been replaced by the amino acid K (lysine), in module C^(C) (SEQID NO: 31) the amino acid S at position 25 has been replaced by theamino acid 25 by C, in module C^(K1) (SEQ ID NO: 32) the amino acid S atposition 25 has been replaced by the amino acid K, in module C^(K2) (SEQID NO: 33) the amino acid E (glutamate) at position 20 has been replacedby the amino acid K, and in module C^(KC) (SEQ ID NO: 34) the amino acidE at position 20 has been replaced by the amino acid K and the aminoacid S at position 25 has been replaced by the amino acid C (WO2007/025719).

It is also preferred that the silk polypeptide according to the presentinvention comprises, essentially consists of, or consists of between 2to 80 repetitive units, between 3 to 80 repetitive units, or between 4to 60 repetitive units, preferably between 8 to 48 repetitive units, orbetween 10 to 40 repetitive units and most preferably between 16 to 32repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units, which areindependently selected from module A^(C) (SEQ ID NO: 29), module A^(K)(SEQ ID NO: 30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO:32), module C^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34). Itshould be noted that at least two of the repetitive units comprised inthe silk polypeptide according to the present invention are identicalrepetitive units (modules).

For example, the silk polypeptide used in the method of the presentinvention can comprises or consists of the modules C^(C) ₄, C^(C) ₈,C^(C) ₁₆, C^(C) ₃₂, A^(C) ₅, A^(C) ₈, or A^(C) ₁₀.

The modules A^(K), C^(C), C^(K1), C^(K2) and C^(KC) can also be combinedwith each other, i.e. module (repetitive unit) A^(K) can be combinedwith module (repetitive unit) C^(C) (i.e. combination A^(K)C^(C)),module (repetitive unit) C^(K1) can be combined with module (repetitiveunit) C^(K2) and with module (repetitive unit) C^(KC) (i.e. combinationC^(K1)C^(K2)C^(KC)), etc., under the proviso that the silk polypeptideused in the method of the present invention comprises or consists of atleast two repetitive units which are identical. Thus, the silkpolypeptide used in the method of the present invention can alsocomprise or consist of the modules (A^(K))_(n), (C^(C))_(n),(C^(K1))_(n), (C^(K2))_(n), (C^(KC))_(n), (A^(K)A^(C))_(n),(C^(C)C^(C))_(n), (C^(K1)C^(K2))_(n), (C^(K2)C^(K1))_(n),(C^(K1)C^(K2)C^(K1))_(n), (C^(K2)C^(K1)C^(K2))_(n),(C^(K1)C^(K2)C^(KC))_(n), (C^(KC)C^(K2)C^(KC))_(n), or(C^(KC)C^(K2)C^(K1))_(n), wherein n is at least 2, preferably 4, 5, 6,7, 8, 10, 12, 16, or 20. The term “combined with each other” is definedabove.

In further preferred embodiments, the repetitive units of the respectivesilk polypeptide, preferably spider silk polypeptide, are independentlyselected from module A (SEQ ID NO: 20) or variants thereof, module C(SEQ ID NO: 21) or variants thereof, module Q (SEQ ID NO: 22) orvariants thereof, module K (SEQ ID NO: 23) or variants thereof, modulesp (SEQ ID NO: 24) or variants thereof, module S (SEQ ID NO: 25) orvariants thereof, module R (SEQ ID NO: 26) or variants thereof, module X(SEQ ID NO: 27) or variants thereof, module Y (SEQ ID NO: 28) orvariants thereof, module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).

In more preferred embodiments, the silk polypeptide according to thepresent invention comprises, essentially consists of, or consists ofbetween 2 to 80, between 3 to 80 repetitive units, or between 4 to 60repetitive units, preferably between 8 to 48 repetitive units, orbetween 10 to 40 repetitive units and most preferably between 16 to 32repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units, which areindependently selected from module A (SEQ ID NO: 20) or variantsthereof, module C (SEQ ID NO: 21) or variants thereof, module Q (SEQ IDNO: 22) or variants thereof, module K (SEQ ID NO: 23) or variantsthereof, module sp (SEQ ID NO: 24) or variants thereof, module S (SEQ IDNO: 25) or variants thereof, module R (SEQ ID NO: 26) or variantsthereof, module X (SEQ ID NO: 27) or variants thereof, module Y (SEQ IDNO: 28) or variants thereof, module A^(C) (SEQ ID NO: 29), module A^(K)(SEQ ID NO: 30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO:32), module C^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).Again, it should be noted that at least two of the repetitive unitscomprised in the silk polypeptide according to the present invention areidentical repetitive units (modules).

The modules A^(K), C^(C), C^(K1), C^(K2) and C^(KC) can also be combinedwith the modules A, C, Q, K, sp, S, R, X or Y, i.e. module (repetitiveunit) A^(K) can be combined with module (repetitive unit) C (i.e.combination A^(K)C), or module (repetitive unit) C^(C) can be combinedwith module (repetitive unit) C (i.e. combination C^(C)C), etc., underthe proviso that the silk polypeptide used in the method of the presentinvention comprises or consists of at least two repetitive units whichare identical. Thus, the silk polypeptide used in the method of thepresent invention can also comprise or consist of the modules(AQA^(K))_(n), (QA^(K))_(n), (QA^(K)Q)_(n), (A^(K)QA)_(n),(A^(K)QA^(K))_(n), (CC^(C))_(n), (CC^(C)C)_(n), (C^(C)C^(C)C)_(n),(CC^(C)C^(C))_(n), (C^(C)Q)_(n), (QC^(C))_(n), (QC^(C)Q)_(n),(C^(C)QC)_(n), (CQC^(C))_(n), (C^(C)QC^(C))_(n), (CC^(K1))_(n),(C^(K1)C)_(n), (C^(K1)CC)_(n), (CC^(K1)C)_(n), (C^(KC)C^(KC)C)_(n),(CC^(KC)C^(KC))_(n), (C^(KC)Q)_(n), (QC^(KC))_(n), (QC^(KC)Q)_(n),(A^(K)C^(K1)Q)_(n), (QC^(K2)A^(K))_(n), or (C^(K1)C^(K2)C)_(n), whereinn is at least 2, preferably 4, 5, 6, 7, 8, 10, 12, 16, or 20. The term“combined with each other” is defined above.

For example, the silk polypeptide used in the method of the presentinvention comprises or consists of the modules C₁₆C^(C), C^(C)C₁₆,C₈C^(C)C₈, C₈C^(C) ₈, C^(C) ₈C₈, C₄C^(C) ₈C₄, C^(C) ₄C₈C^(C) ₄,C^(C)(AQ)₂₄, or (AQ)₂₄C^(C).

The term “independently selected” as used herein means that the silkpolypeptide, e.g. spider silk polypeptide, may comprise one or moredifferent repetitive units each comprising one or more of the abovedescribed modules. As mentioned above, the silk polypeptides, e.g.spider silk polypeptides, according to the invention comprise at leasttwo identical repetitive units.

The term “variants thereof” as used herein means that suitable aminoacid sequences are not necessarily restricted to the exact sequences asgiven in the SEQ ID NOs. Variants of the amino acid sequences indicatedherein may also comprise sequences wherein one or more amino acid areinserted, deleted, modified and/or substituted.

Variants of the amino acid sequences as described herein are capable ofproducing polypeptides having the same properties, i.e. having the sameor similar secondary structural elements. Preferably not more than 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, or 20%, more preferably not more than 15%, even morepreferably not more than 10%, most preferably not more than 5% or 2% ofall amino acids of the polypeptide are altered (i.e. are deleted,inserted, modified and/or substituted).

Preferably, in all these embodiments the sequence identity is at leastabout 80%, 85% or 90%, more preferably at least about 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, and most preferably at least about 99%.Sequence identity may be determined over the whole length of therespective sequences.

The determination of percent identity between two sequences ispreferably accomplished using the mathematical algorithm of Karlin andAltschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Such analgorithm is incorporated into the BLASTn and BLASTp programs ofAltschul et al. (1990) J. Mol. Biol. 215: 403-410 available at NCBI(http://www.ncbi.nlm.nih.gov/blast/Blast.cge).

The determination of percent identity is performed with the standardparameters of the BLASTn and BLASTp programs.

BLAST polynucleotide searches are performed with the BLASTn program.

For the general parameters, the “Max Target Sequences” box may be set to100, the “Short queries” box may be ticked, the “Expect threshold” boxmay be set to 10 and the “Word Size” box may be set to 28. For thescoring parameters the “Match/mismatch Scores” may be set to 1,−2 andthe “Gap Costs” box may be set to linear. For the Filters and Maskingparameters, the “Low complexity regions” box may not be ticked, the“Species-specific repeats” box may not be ticked, the “Mask for lookuptable only” box may be ticked, the “Mask lower case letters” box may notbe ticked.

BLAST protein searches are performed with the BLASTp program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to “3”. For the scoringparameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs”Box may be set to “Existence: 11 Extension:1”, the “Compositionaladjustments” box may be set to “Conditional compositional score matrixadjustment”. For the Filters and Masking parameters the “Low complexityregions” box may not be ticked, the “Mask for lookup table only” box maynot be ticked and the “Mask lower case letters” box may not be ticked.

By “modification” it is meant that amino acids of the polypeptide may bechemically or biologically modified, e.g. by glycosylation, amidation,phosphorylation, ubiquitination, etc.

“Substitution” is the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, i.e.conservative amino acid replacements. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity and/or hydrophilicity of certain residues of the aminoacid sequence. Examples of preferred suitable amino acid substitutionsare given in the table below:

Original radical Examples of substitution Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val LeuIle; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr ThrSer Trp Tyr Tyr Trp; Phe Val Ile; Leu

“Insertions” or “deletions” typically can be in the range of about 1 to5 amino acids, preferably about 1, 2 or 3 amino acids. Amino acidadditions are typically not more than 100, preferably not more than 80,more preferably not more than 50, most preferably not more than 20 aminoacids, which are added and/or inserted into the proteins. Further, onlythose additions are contemplated which do not negatively affect thedesired characteristics of the proteins.

Particularly, a module A, C, Q, K, sp, S, R, X or Y variant differs fromthe reference (wild-type) module A, C, Q, K, sp, S, R, X or Y from whichit is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 amino acid changes in the amino acid sequence (i.e. substitutions,additions, insertions, deletions, N-terminal truncations and/orC-terminal truncations). Such a module variant can alternatively oradditionally be characterized by a certain degree of sequence identityto the reference (wild-type) module from which it is derived. Thus, amodule A, C, Q, K, sp, S, R, X or Y variant has a sequence identity ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 99.9% to therespective reference (wild-type) module A, C, Q, K, sp, S, R, X or Y.Preferably, the sequence identity is over a continuous stretch of atleast 10, 15, 18, 20, 24, 27, 28, 30, 34, 35, or more amino acids,preferably over the whole length of the respective reference (wild-type)module A, C, Q, K, sp, S, R, X or Y.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference (wild-type) module A, C, Q, K, sp, S,R, X or Y. It is further particularly preferred that the sequenceidentity is at least 80% over a continuous stretch of at least 10, 15,18, 20, 24, 28, or 30 amino acids, is at least 85% over a continuousstretch of at least 10, 15, 18, 20, 24, 28, or 30 amino acids, is atleast 90% over a continuous stretch of at least 10, 15, 18, 20, 24, 28,or 30 amino acids, is at least 95% over a continuous stretch of at least10, 15, 18, 20, 24, 28, or 30 amino acids, is at least 98% over acontinuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 aminoacids, or is at least 99% over a continuous stretch of at least 10, 15,18, 20, 24, 28, or 30 amino acids of the respective reference(wild-type) module A, C, Q, K, sp, S, R, X or Y.

A fragment (or deletion variant) of module A, C, Q, K, sp, S, R, X or Yhas preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or 15 amino acids at its N-terminus and/or at itsC-terminus. The deletion can also be internally.

Additionally, the module A, C, Q, K, sp, S, R, X or Y variant orfragment is only regarded as a module A, C, Q, K, sp, S, R, X or Yvariant or fragment within the context of the present invention, if thechanges with respect to the amino acid sequence on which the variant orfragment is based do not negatively affect the ability of the silkparticle comprising the silk polypeptide to be loaded with a compound.Preferably, the silk particle comprising the silk polypeptide whichcomprises the module A, C, Q, K, sp, S, R, X, or Y variant or fragmentis capable of being loaded with a compound so that at least 20%,preferably at least 40%, more preferably at least 50%, 60%, 70%, 80%,90%, or 95% of the loaded compound is located within the matrix of thesilk particle. The skilled person can readily determine the “loading” ofa silk particle (e.g. via UV-Vis-spectroscopy), in particular thepercentage of the compound which is located within the matrix of silkparticles (see, for example, experimental section).

As mentioned above, the silk polypeptide, e.g. spider silk polypeptide,may be an authentic polypeptide naturally occurring in nature or besynthetically or recombinantly produced. When recombinantly produced,the above described modules and sequences may be combined to yield thesilk polypeptide, e.g. spider silk polypeptide, with favourablecharacteristics. Preferably, the modules may be combined such that theresulting polypeptide possesses at least two identical repetitive units.

In specific embodiments, the silk polypeptide, preferably spider silkpolypeptide, further comprises at least one non-repetitive (NR) unit,i.e. 1, 2, 3, 4, 5, 6, or more NR units, preferably one NR unit.Preferably, the NR sequences are authentic sequences.

In the context of the present invention, the term “non-repetitive (NR)unit” refers to a region of amino acids present in a naturally occurringsilk polypeptide that displays no obvious repetition pattern(non-repetitive unit or NR unit).

Preferably, the amino acid sequence of the non-repetitive unitcorresponds to a non-repetitive amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1 orSEQ ID NO: 47) or ADF-4 (SEQ ID NO: 2 or SEQ ID NO: 48), or to an aminoacid sequence substantially similar thereto.

It is particularly preferred that the amino acid sequence of thenon-repetitive unit corresponds to a non-repetitive carboxy terminalamino acid sequence of naturally occurring dragline polypeptides,preferably of ADF-3 (SEQ ID NO: 1 or SEQ ID NO: 47) or ADF-4 (SEQ ID NO:2 or SEQ ID NO: 48), or to an amino acid sequence substantially similarthereto. More preferably, the amino acid sequence of the non-repetitiveunit corresponds to a non-repetitive carboxy terminal amino acidsequence of ADF-3 (SEQ ID NO: 1) which comprises amino acids 513 through636, or of ADF-4 (SEQ ID NO: 2) which comprises amino acids 302 through410, or to an amino acid sequence substantially similar thereto.

In this regard “substantially similar” means a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over 20, 30, 40, 50, 60, 70, 80 or more amino acids,more preferably over the whole length of the respective referencenon-repetitive (carboxy terminal) amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2).

A “non-repetitive unit” having an amino acid sequence which is“substantially similar” to a corresponding non-repetitive (carboxyterminal) amino acid sequence within a naturally occurring draglinepolypeptide (i.e. wild-type non-repetitive (carboxy terminal) unit),preferably within ADF-3 (SEQ ID NO: 1 or SEQ ID NO: 47) or ADF-4 (SEQ IDNO: 2 or SEQ ID NO: 48), is also similar with respect to its functionalproperties, e.g. the silk particle comprising the silk polypeptide whichcomprises the “substantially similar non-repetitive unit” can still beloaded with a compound. Preferably, the silk particle comprising thesilk polypeptide which comprises the “substantially similarnon-repetitive unit” is capable of being loaded with a compound so thatat least 20%, preferably at least 40%, more preferably at least 50%,60%, 70%, 80%, 90%, or 95% of the loaded compound is located within thematrix of the silk particle. The skilled person can readily determinethe “loading” of a silk particle (e.g. via UV-Vis-spectroscopy), inparticular the percentage of the compound which is located within thematrix of silk particles (see, for example, experimental section).

More preferably, the non-repetitive (NR) unit is independently selectedfrom the group consisting of NR3 (SEQ ID NO: 41 and SEQ ID NO: 45) orvariants thereof and NR4 (SEQ ID NO: 42 and SEQ ID NO: 46) or variantsthereof. The NR3 (SEQ ID NO: 41) unit is based on the amino acidsequence of ADF-3 of the spider Araneus diadematus and the NR4 (SEQ IDNO: 42) unit is based on the amino acid sequence of ADF-4 of the spiderAraneus diadematus (WO 2006/008163).

A NR3 or NR4 unit variant differs from the reference NR3 (SEQ ID NO: 41or SEQ ID NO: 45) or NR4 (SEQ ID NO: 42 or SEQ ID NO: 46) unit fromwhich it is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, or 30 amino acid changes in the aminoacid sequence (i.e. exchanges, insertions, deletions, N-terminaltruncations and/or C-terminal truncations). Such a NR3 or NR4 unitvariant can alternatively or additionally be characterized by a certaindegree of sequence identity to the reference NR3 or NR4 unit from whichit is derived. Thus, a NR3 or NR4 unit variant has a sequence identityof at least 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or even 99.9% to the respective reference NR3 or NR4unit. Preferably, the sequence identity is over a continuous stretch ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acids,preferably over the whole length of the respective reference NR3 or NR4unit.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference NR3 or NR4 unit. It is furtherparticularly preferred that the sequence identity is at least 80% over acontinuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 aminoacids, is at least 85% over a continuous stretch of at least 20, 30, 40,50, 60, 70, or 80 amino acids, is at least 90% over a continuous stretchof at least 20, 30, 40, 50, 60, 70, or 80 amino acids, is at least 95%over a continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80amino acids, is at least 98% over a continuous stretch of at least 20,30, 40, 50, 60, 70, or 80 amino acids, or is at least 99% over acontinuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 amino acidsof the respective reference NR3 or NR4 unit.

A fragment (or deletion variant) of a NR3 or NR4 unit has preferably adeletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 aminoacids at its N-terminus and/or at its C-terminus. The deletion can alsobe internally.

Additionally, the NR3 or NR4 unit variant or fragment is only regardedas a NR3 or NR4 unit variant or fragment within the context of thepresent invention, if the changes with respect to the amino acidsequence on which the variant or fragment is based do not negativelyaffect the ability of the silk particle comprising the silk polypeptideto be loaded with a compound. Preferably, the silk particle comprisingthe silk polypeptide which comprises the NR3 or NR4 unit variant orfragment is capable of being loaded with a compound so that at least20%, preferably at least 40%, more preferably at least 50%, 60%, 70%,80%, 90%, or 95% of the loaded compound is located within the matrix ofthe silk particle. The skilled person can readily determine the“loading” of a silk particle (e.g. via UV-Vis-spectroscopy), inparticular the percentage of the compound which is located within thematrix of silk particles (see, for example, experimental section).Within the context of the present invention, the term “authentic” meansthat the respective nucleic acid sequences are isolated from theirnatural environment without substantial modifications being made to thesequence itself. However, this does not mean that the nucleic acidsequences may not be modified in order to adapt the sequence to theexpression in a specific host without changing the resulting amino acidsequence encoded therefrom (codon usage adaption).

In further specific embodiments, the silk polypeptide, preferably spidersilk polypeptide, is selected from the group consisting of ADF-3 (SEQ IDNO: 1 and SEQ ID NO: 47) or variants thereof, ADF-4 (SEQ ID NO: 2 andSEQ ID NO: 48) or variants thereof, MaSp I (SEQ ID NO: 43 and SEQ IDNOs: 53-64) or variants thereof, MaSp II (SEQ ID NO: 44 and SEQ ID NOs:65-78) or variants thereof, (C)_(m)NR_(z), NR_(z)(C)_(m),(AQ)_(n)NR_(z), NR_(z)(AQ)_(n), NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z),(C)_(m), (AQ)_(n), (QAQ)_(o), Y_(p), X_(p), and IC_(P), wherein m is aninteger of 8 to 48 (i.e. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48), n is an integer of 6 to24 (i.e. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24), o is an integer of 8 to 16 (i.e. 8, 9, 10, 11, 12, 13, 14,15, or 16), p is an integer of 8 to 16 (i.e. 8, 9, 10, 11, 12, 13, 14,15, or 16), z is an integer of 1 to (i.e. 1, 2, or 3), and NR stands fora non-repetitive unit. The above mentioned formulas are defined by oneof the following: In the formula

-   -   (i) (C)_(m), a “m” number of C modules, namely 8 to 48 C        modules, represented by the amino acid sequence according to SEQ        ID NO: 21, are combined with each other,    -   (ii) (C)_(m)NR_(z), a “m” number of C modules, namely 8 to 48 C        modules, represented by the amino acid sequence according to SEQ        ID NO: 21, are combined with each other, wherein said C modules        are further combined with a “z” number of non-repetitive (NR)        units, namely 1 to 3 non-repetitive (NR) units, e.g. the        non-repetitive (NR) units NR3 represented by the amino acid        sequence according to SEQ ID NO: 41 or NR4 represented by the        amino acid sequence according to SEQ ID NO: 42,    -   (iii) NR_(z)(C)_(m), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41 or NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, is present (z=1) or are combined        with each other (z=2 or 3), wherein said non-repetitive (NR)        unit(s) is (are) further combined with a “m” number of C        modules, namely 8 to 48 C modules, represented by the amino acid        sequence according to SEQ ID NO: 21,    -   (iv) (AQ)_(n), a “n” number of A and Q module combinations,        namely 6 to 24 A and Q module combinations, wherein module A is        represented by the amino acid sequence according to SEQ ID NO:        20 and module Q is represented by the amino acid sequence        according to SEQ ID NO: 22, are combined with each other,    -   (v) (AQ)_(n)NR_(z), a “n” number of A and Q module combinations,        namely 6 to 24 A and Q module combinations, wherein module A is        represented by the amino acid sequence according to SEQ ID NO:        20 and module Q is represented by the amino acid sequence        according to SEQ ID NO: 22, are combined with each other, and        wherein said A and Q module combinations are further combined        with a “z” number of non-repetitive (NR) units, namely 1 to 3        non-repetitive (NR) units, e.g. the non-repetitive (NR) units        NR3 represented by the amino acid sequence according to SEQ ID        NO: 41 or NR4 represented by the amino acid sequence according        to SEQ ID NO: 42,    -   (vi) NR_(z)(AQ)_(n), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41 or NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, is present (z=1) or are combined        with each other (z=2 or 3), wherein said non-repetitive (NR)        unit(s) is (are) further combined with a “n” number of A and Q        module combinations, namely 6 to 24 A and Q module combinations,        wherein module A is represented by the amino acid sequence        according to SEQ ID NO: 20 and module Q is represented by the        amino acid sequence according to SEQ ID NO: 22,    -   (vii) (QAQ)_(o), a “o” number of Q, A and Q module combinations,        namely 8 to 16 Q, A and Q module combinations, wherein module Q        is represented by an amino acid sequence according to SEQ ID NO:        22 and module A is represented by the amino acid sequence        according to SEQ ID NO: 20, are combined with each other,    -   (viii) (QAQ)_(o)NR_(z), a “o” number of Q, A and Q module        combinations, namely 8 to 16 Q, A and Q module combinations,        wherein module Q is represented by an amino acid sequence        according to SEQ ID NO: 22 and module A is represented by the        amino acid sequence according to SEQ ID NO: 20, are combined        with each other, and wherein said Q, A and Q module combinations        are further combined with a “z” number of non-repetitive (NR)        units, namely 1 to 3 non-repetitive (NR) units, e.g. the        non-repetitive (NR) units NR3 represented by the amino acid        sequence according to SEQ ID NO: 41 or NR4 represented by the        amino acid sequence according to SEQ ID NO: 42,    -   (ix) NR_(z)(QAQ)_(o), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41 or NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, is present (z=1) or are combined        with each other (z=2 or 3), wherein said non-repetitive (NR)        unit(s) is (are) further combined with a “o” number of Q, A and        Q module combinations, namely 8 to 16 Q, A and Q module        combinations, wherein module Q is represented by an amino acid        sequence according to SEQ ID NO: 22 and module A is represented        by the amino acid sequence according to SEQ ID NO: 20,    -   (x) Y_(p), a “p” number of Y modules, namely 8 to 16 Y modules,        represented by the amino acid sequence according to SEQ ID NO:        28, are combined with each other,    -   (xi) X_(p), a “p” number of X modules, namely 8 to 16 X modules,        represented by the amino acid sequence according to SEQ ID NO:        27, are combined with each other, and    -   (xii) K_(p), a “p” number of K modules, namely 8 to 16 K        modules, represented by the amino acid sequence according to SEQ        ID NO: 23, are combined with each other.

More preferably, the silk polypeptide, preferably spider silkpolypeptide, is C₁₆, C₃₂, (AQ)₁₂, (AQ)₂₄, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3,(AQ)₂₄NR3, Y₈, Y₁₆, X₈, X₁₆, K₈, or K₁₆.

An ADF-3, ADF-4, MaSp I or MaSp II variant differs from the reference(wild-type) ADF-3 (SEQ ID NO: 1 or SEQ ID NO: 47), ADF-4 (SEQ ID NO: 2or SEQ ID NO: 48), MaSp I (SEQ ID NO: 43 and SEQ ID NOs: 53 to 64) orMaSp II (SEQ ID NO: 44 and SEQ ID NOs: 65 to 78) polypeptide from whichit is derived by up to 150 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150) amino acid changesin the amino acid sequence (i.e. substitutions, insertions, deletions,N-terminal truncations and/or C-terminal truncations). Such a variantcan alternatively or additionally be characterized by a certain degreeof sequence identity to the reference (wild-type) polypeptide from whichit is derived. Thus, an ADF-3, ADF-4, MaSp I or MaSp II variant has asequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 99.9% to the respective reference (wild-type) ADF-3, ADF-4, MaSp Ior MaSp II polypeptide. Preferably, the sequence identity is over acontinuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 120, 150, 180, 200, 250, 300, 350, 400, or more amino acids,preferably over the whole length of the respective reference (wild-type)ADF-3, ADF-4, MaSp I or MaSp II polypeptide.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference (wild-type) ADF-3, ADF-4, MaSp I orMaSp II polypeptide. It is further particularly preferred that thesequence identity is at least 80% over a continuous stretch of at least20, 30, 50, 100, 150, 200, 250, or 300 amino acids, is at least 85% overa continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids, is at least 90% over a continuous stretch of at least 20,30, 50, 100, 150, 200, 250, or 300 amino acids, is at least 95% over acontinuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids, is at least 98% over a continuous stretch of at least 20,30, 50, 100, 150, 200, 250, or 300 amino acids, or is at least 99% overa continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids of the respective reference (wild-type) ADF-3, ADF-4, MaSp Ior MaSp II polypeptide.

A fragment (or deletion variant) of the ADF-3 (SEQ ID NO: 1) polypeptidehas preferably a deletion of up to 1, 2, 3, 4; S, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,120, 150, 170, 200, 220, 250, 270, 300, 320, 350, 370, 400, 420, 450,470, 500, 520, 550, 570, 600, or 610 amino acids at its N-terminusand/or at its C-terminus. The deletion can also be internally.

A fragment (or deletion variant) of the ADF-4 (SEQ ID NO: 2) polypeptidehas preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,120, 150, 170, 200, 220, 250, 270, 300, 320, 330, 340, 350, 360, 370,380, or 390 amino acids at its N-terminus and/or at its C-terminus. Thedeletion can also be internally.

A fragment (or deletion variant) of the MaSp I (SEQ ID NO: 43)polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 620, 640,660, 670, 680, or 690 amino acids at its N-terminus and/or at itsC-terminus. The deletion can also be internally.

A fragment (or deletion variant) of the MaSp II (SEQ ID NO: 44)polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520, 540, 560, or 570amino acids at its N-terminus and/or at its C-terminus. The deletion canalso be internally.

Additionally, the ADF-3, ADF-4, MaSp I or MaSp II variant or fragment isonly regarded as an ADF-3, ADF-4, MaSp I or MaSp II variant or fragmentwithin the context of the present invention, if the changes with respectto the amino acid sequence on which the variant or fragment is based donot negatively affect the ability of the silk particle comprising thesilk polypeptide to be loaded with a compound. Preferably, the silkparticle comprising the silk polypeptide which comprises the ADF-3,ADF-4, MaSp I or MaSp II variant or fragment is capable of being loadedwith a compound so that at least 20%, preferably at least 40%, morepreferably at least 50%, 60%, 70%, 80%, 90%, or 95% of the loadedcompound is located within the matrix of the silk particle. The skilledperson can readily determine the “loading” of a silk particle (e.g. viaUV-Vis-spectroscopy), in particular the percentage of the compound whichis located within the matrix of silk particles (see, for example,experimental section).

Preferably, the concentration of the silk polypeptide, more preferablyspider silk polypeptide, in the aqueous solution is of between 0.01 wt%/vol and 30 wt %/vol, more preferably 0.1 wt %/vol and 30 wt %/vol, andmost preferably between 1 wt %/vol and 20 wt %/vol, e.g. 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 wt %/vol.

In preferred embodiments, aggregation is triggered by pH-shift, ionexchange, shear forces, the addition of an alcohol or a lyotropic saltor by a combination thereof. More preferably, the pH-shift is achievedby lowering the pH of the aqueous silk solution, preferably spider silksolution. Even more preferred is a pH of less than 4, less than 3, lessthan 2 and most preferred of about 1. Preferred alcohols for triggeringaggregation are selected from the group consisting of methanol, ethanol,and isopropanol. In a preferred embodiment, the alcohol is methanol.Preferably, aggregation may be triggered by the addition of ions, whichgenerally leads to the salting-out of proteins. In particular,structural formation of the unfolded proteins may thereby be induced.The salting out-properties of ions are generally described by theHofmeister series. The “Hofmeister series” or “lyotropic series” is aclassification of ions in order of their ability to change waterstructure. The effects of these changes were first worked out by FranzHofmeister, who studied the effects of cations and anions on thesolubility of proteins. Thereafter, anions appear to have a largereffect than cations, and are usually ordered F⁻=SO₄ ²⁻>HPO₄²⁻>acetate>Cl⁻>NO₃ ⁻>Br⁻>ClO₃ ⁻>I⁻>ClO₄ ⁻. The order of cations isusually given as NH₄ ⁺>K⁺>Na⁺>Li⁺>Mg²⁺>Ca²⁺>guanidinium. Generally anylyotropic salt can be used to trigger aggregation of silk polypeptides,e.g. spider silk polypeptides. Preferred lyotropic salts which can beused to trigger aggregation are selected from the group consisting ofammonium sulphate, sodium phosphate, potassium phosphate and carbonatesalts such as ammonium carbonate, sodium carbonate or potassiumcarbonate. In further preferred embodiments the lyotropic salt isselected from the group consisting of ammonium sulphate, sodiumphosphate, and potassium phosphate. Preferably, the concentration of thelyotropic salt is of between about 400 mM and about 3 M, preferablyabout 1 to about 2 M, most preferably about 2 M, e.g. 400 mM, 500 mM,600 mM, 700 mM, 800 mM, 900 mM, 1 M, 1.5 M, 2 M, 2.5 M, or 3 M.

In preferred embodiments of the invention, the compound is apharmaceutically active compound, a cosmetic substance, an agriculturalsubstance, a chemoattractant, a chemorepellent, an anti-fungalsubstance, an anti-bacterial substance, a nutrient, a dietarysupplement, a dye, a fragrance or an agent selected from the groupconsisting of hemostatic agents, growth stimulating agents, inflammatoryagents, anti-fouling agents, antimicrobial agents and UV protectingagents.

Preferably, the compound has an overall positive net charge. The terms“positive charge” and “cationic” can be used interchangeably. As will beshown in detail in the examples, especially positively charged compoundsare well-suited for the loading of silk particles, e.g. spider silkparticles. As used herein, “positive charge” means that the compoundpossesses at least one elementary charge of a proton. The skilled personknows that the presence of at least one charge of a water-solublecompound is dependent on factors such as the pK_(a)-value of thecompound and the pH of the aqueous solvent.

As used herein, the term “pK_(a)-value”, (also known as acidityconstant, or acid-ionization constant) is a quantitative measure of thestrength of an acid in solution. It is derived from the dissociationconstant K_(a) which describes the equilibrium for a chemical reactionknown as dissociation in the context of an acid-base reaction. Due tothe many orders of magnitude spanned by K_(a) values, a logarithmicmeasure of the acid dissociation constant is more commonly used inpractice. The larger the value pK_(a) the smaller the extent ofdissociation and the less strong is an acid. Accordingly, the pK_(b)value describes the strength of a base in solution.

In aqueous solutions the pK_(a)-value may give an indication whether acompound has a positive charge or not. Preferably the compound possessesa positive net charge at the pH used for the loading step.

Various other methods for determining or measuring the net charge of acompound are known to one of skill in the art. For example, the netcharge can typically be measured using electrophoretic methods. Thecharge of a molecule in aqueous solution may also be predicted usingsuitable software such as ACD/ChemSketch (available at AdvancedChemistry Development, ACD/labs, http://www.acdlabs.com).

The person skilled in the art also knows how to determine whichcompounds are suitable for loading, i.e. whether a compound of interestpossesses at least one positive charge at the pH of the aqueous solutionused for loading the particles.

As will be clear from the description below and in the examples, methodsfor assessing whether a compound is suitable for loading of the silkparticles, e.g. spider silk particles, according to the inventioninclude titration methods and the measurement of the zeta-potentialduring titration.

If the compound is a peptide or a protein or any other amphiphiliccompound, the presence of an overall positive net charge is dependent onthe isoelectric point (pI) value of the compound. The isoelectric point,sometimes abbreviated IEP, is the pH at which a particular molecule orsurface carries no net electrical charge. For example, amphotericmolecules or zwitterions contain both positive and negative chargesdepending on the functional groups present in the molecule. The netcharge on the molecule is affected by pH of their surroundingenvironment and can become more positively or negatively charged due tothe loss or gain of protons. The pI the pH value at which the moleculecarries no electrical charge or the negative and positive charge areequal.

Methods for determining whether a peptide at a certain pH has apredominant net charge are known in the art. For example, suitable toolsfor calculating the pI value of proteins or peptides are provided byExPasyProteomic server (www.expasy.ch). The program “Compute pI/Mw” is atool which allows the computation of the theoretical pI (isoelectricpoint) and Mw (molecular weight) for a list of database entries(UniProtKnowledgebase (Swiss-prot or TrEMBL)) or for user enteredsequences. Prediction of pI values are also described in Bjellqvist etal. (1993) and Gasteider et al. (2005) [Bjellqvist, B., The focusingpositions of polypeptides in immobilized pH gradients can be predictedfrom their amino acid sequences. Electrophoresis 1993, 14, 1023-1031.Gasteiger E., Protein Identification and Analysis Tools on the ExPASyServer, (In) John M. Walker (ed): The Proteomics Protocols Handbook,Humana Press (2005).]

In further specific embodiments, the compound is able to permeate intothe silk matrix, preferably spider silk matrix, by electrostaticinteraction and/or diffusion. It has to be understood that the chargedcompound is attracted by an overall negative net charge of the silkparticles, e.g. spider silk particles. Due to the attraction based onthe presence of opposite charges, the compound is capable of adhering tothe surface of the silk particle, e.g. spider silk particle, anddiffusing into the silk matrix, e.g. spider silk matrix. Generally therepulsion or attraction of charges in colloidal systems can be explainedwith the zeta potential. Within the context of the present invention, apositively charged compound is of permeating into the silk matrix, e.g.spider silk matrix, by electrostatic interaction when the zeta potentialof the silk particles, e.g. spider silk particles, is essentiallynegative.

Naturally, the one or more silk polypeptides, e.g. spider silkpolypeptides, of the silk particles, e.g. spider silk particles, possessat least one negative charge at the carboxyl terminus. As used herein,the terms “negatively charged” and “anionic” can be usedinterchangeably. The person skilled in the art also knows how to selectappropriate amino acid sequences in order obtain a polypeptide having anoverall negative net charge. For example, this can be achieved byselecting sequences comprising negatively charged amino acids. Asuitable negatively charged silk polypeptide, particularly spider silkpolypeptide, is for example C₁₆ which comprises 16 repeats of thesequence of module C (SEQ ID NO: 21) or variants thereof. In preferredembodiments, the compound has a neutral or alkaline nature.

As used herein, the terms “pharmaceutical active compound”, “drug”,“pharmaceutical agent”, “therapeutic agent” or “bioactivecompound/agent” may be used interchangeably and refer to any physical,chemical or biological substance which may be used in the treatment,cure, prophylaxis, prevention, or diagnosis of a pathological condition,e.g. a disease or disorder, or which may be used to otherwise enhancephysical, psychical or mental well-being. Accordingly, pharmaceuticallyactive compounds envisaged in the context of the present inventioninclude any compound with therapeutic or prophylactic effects. Forexample, it can be a compound that affects or participates in tissuegrowth, cell growth, cell differentiation, a compound that is able toinvoke a biological action such as an immune response, or a compoundthat can play any other role in one or more biological processes.

The therapeutic agent can be, but is not limited to, an antimicrobialagent, an antibiotic, an anti-viral agent, anti-fungal agent, an urinarytract antiseptic, an agent for treating anaerobic infections, an agentfor treating tuberculosis, an agent for treating leprosy, an agent fortreating amebiasis, an anti-malarial agent, an anti-helminthiasis agent,an anti-gout agent, a thrombin inhibitors, an antithrombogenic agent, athrombolytic agent, fibrinolytic agent, a vasospasm inhibitor, avasodilator, an antihypertensive agent, an antihypotensive agent, aninhibitors of surface glycoprotein receptor, antiplatelet agent, anantimitotic, an actin inhibitors, a microtubule inhibitor, an antisecretory agent, a remodeling inhibitor, an antimetabolite, anantiproliferative (including anti-angiogenesis agents), animmunosuppressive agents, a growth hormone antagonist, a growth factor,a dopamine agonist, a radiotherapeutic agent, a extracellular matrixcomponent, an ACE inhibitor, a free radical scavenger, a chelator, anantioxidant, an antipolymerase, a photodynamic therapy agent, acentrally active muscle relaxant, an opioid agonist, a non-opioidanalgesic, a non-steroid anti-inflammatory agent, an antimigraine agent,a Cox-II inhibitor, an antiemetic, a β-adrenergic blocker, aCa²⁺-channel blocker, an anticonvulsant, an antidepressant, ananticancer agent, an agent for treating or preventing urinaryincontinence (UI), an agent for treating or preventing an ulcer, anagent for treating or preventing infectious bursal disease (IBD), anagent for treating or preventing irritable bowel syndrome (IBS), anagent for treating addictive disorder, an agent for treating Parkinson'sdisease and parkinsonism, an agent for treating anxiety, an agent fortreating epilepsy, an agent for treating a stroke, an agent for treatinga seizure, an agent for treating a pruritic condition, an agent fortreating psychosis, an agent for treating Huntington's chorea, an agentfor treating amytrophic lateral sclerosis (ALS), an agent for treating acognitive disorder, an agent for treating a migraine, an agent fortreating vomiting, an agent for treating dyskinesia, or an agent fortreating depression, an anorexic, an antacid, antiacne agents, anantiallergic, an antianginal agent, an antiarrythmic, an antiasthmatic,an antibaldness agent, anticholinergic agent, an anticoagulant and bloodthinner, anticolitis agent, an anticystitis agent, an antidiabeticagent, an antidiarrheal, an antidiuretic, an antiflatulent, anantiglaucoma agent, an antihistaminic, an antipneumonia agent, anantiobesity agent, an antipsoriatics, antipsychotic, an antipyretic,antirheumatic, antitussive, a bone densifier, a carbonic anhydraseinhibitor, a cardiotonic, a contraceptive, a decongestant, a diuretic, aCNS stimulant, dopamine receptor antagonist, HMG CoA reductaseinhibitor, a phosphodiesterase inhibitor, a hormone, a hormoneantagonist, a hematopoietic agent, an immunomodulator, animmunosuppressant, a laxative, an agent for treating multiple sclerosis,a sedative, a serotonin uptake inhibitor, and mixtures thereof.

Examples of useful antimicrobial agents belong to, but are not limitedto, the group of antibiotics comprising ampicillin, nafcillin,amoxicillin, oxacillin, azlocillin, penicillin G, carbenicillin,penicillin V, dicloxacillin, phenethicillin, floxacillin, piperacillin,mecillinam, sulbenicillin, methicillin, icarcillin, mezlocillin,cephalosporins such as cefaclor, cephalothin, cefadroxil, cephapirin,cefamandole, cephradine, cefatrizine, cefsulodine, cefazolin,ceftazidim, ceforanide, ceftriaxon, cefoxitin, cefuroxime, cephacetrile,latamoxef, or cephalexin, aminoglycosides such as amikacin, neomycin,dibekacyn, kanamycin, gentamycin, netilmycin or tobramycin, macrolidessuch as amphotericin B, novobiocin, bacitracin, nystatin, clindamycin,polymyxins, colistin, rovamycin, erythromycin, spectinomycin, lincomycinor vancomycin, tetracyclines such as chlortetracycline, oxytetracycline,demeclocycline, rolitetracycline, doxycycline, tetracycline,minocycline, chloramphenicol, rifamycin, rifampicin and thiamphenicol.

Examples of useful antifungal agents belong to, but are not limited to,the group comprising amphotericin B, ketoconazole, clotrimazole,miconazole, econazole, natamycin, flucytosine, nystatine andgriseofulvin.

Examples of useful antiviral agents belong to, but are not limited to,the group comprising aciclovir, idoxuridine, amantidine, methisazone,cytarabine, vidarabine and ganciclovir.

Examples of useful urinary tract antiseptics belong to, but are notlimited to, the group comprising methanamine, quinolones such asnorfloxacin or cinoxacin, nalidixic acid, and nitro-compounds such asnitrofurantoine, nifurtoinol or oxolinic acid.

An example of an agent for treating anaerobic infections belong to, butis not limited to, metronidazole.

Examples of useful therapeutic agents for treating tuberculosis belongto, but are not limited to, the group comprising aminosalicyclic acid,isoniazide, cycloserine, rifampicine, ethambutol, tiocarlide,ethionamide and viomycin.

Examples of useful therapeutic agents for treating leprosy belong to,but are not limited to, the group comprising amithiozone, rifampicine,clofazimine, sodium sulfoxone and diaminodiphenylsulfone (DDS, dapsone).

Examples of useful chemotherapeutics for treatment of amebiasis belongto, but are not limited to, the group comprising chloroquine,iodoquinol, clioquinol, metronidazole, dehydroemetine, paromomycin,diloxanide, furoatetinidazole and emetine.

Examples of useful anti-malarial agents belong to, but are not limitedto, the group comprising chloroquine, pyrimethamine, hydroxychloroquine,quinine, mefloquine, sulfadoxine/pyrimethamine, pentamidine, sodiumsuramin, primaquine, trimethoprim and proguanil.

Examples of useful anti-helminthiasis agents belong to, but are notlimited to, the group comprising antimony potassium tartrate,niridazole, antimony sodium dimercaptosuccinate, oxamniquine, bephenium,piperazine, dichlorophen, praziquantel, diethylcarbamazine, pyrantelparmoate, hycanthone, pyrivium pamoate, levamisole, stibophen,mebendazole, tetramisole, metrifonate, thiobendazole and niclosamide.

Examples of useful anti-gout agents belong to, but are not limited to,the group comprising colchicine and allopurinol.

Examples of useful local anesthetics belong to, but are not limited to,the group comprising articaine, mepivacaine, bupivacaine, prilocalne,etidocaine, procaine, lidocaine or tetracaine.

Examples of useful centrally active muscle relaxants belong to, but arenot limited to, the group comprising baclofen, carisoprodol,chlormezanone, chlorzoxazone, cyclobenzaprine, dantrolene, diazepam,febarbamate, mefenoxalone, mephenesin, metoxalone, methocarbamol ortolperisone.

Examples of useful thyroid drugs in therapy belong to, but are notlimited to, the group comprising levothyronine and liothyronine.

Examples of useful anti-thyroid drugs belong to, but are not limited to,the group comprising carbimazole, methimazole, methylthiouracil andpropylthiouracil.

Examples of useful opioid agonists belong to, but are not limited to,the group comprising alfentanil, allylprodine, alphaprodine,anileridine, benzylmorphine, bezitramide, buprenorphine, butorphanol,clonitazene, codeine, desomorphine, dextromoramide, dezocine,diampromide, diamorphone, dihydrocodeine, dihydromorphine, dimenoxadol,dimepheptanol, dimethylthiambutene, dioxaphetyl butyrate, dipipanone,eptazocine, ethoheptazine, ethylmethylthiambutene, ethylmorphine,etonitazene fentanyl, heroin, hydrocodone, hydromorphone,hydroxypethidine, isomethadone, ketobemidone, levorphanol,levophenacylmorphan, lofentanil, meperidine, meptazinol, metazocine,methadone, metopon, morphine, myrophine, nalbuphine, narceine,nicomorphine, norlevorphanol, normethadone, nalorphine, normorphine,norpipanone, opium, oxycodone, oxymorphone, papavereturn, pentazocine,phenadoxone, phenomorphan, phenazocine, phenoperidine, piminodine,piritramide, proheptazine, promedol, properidine, propiram,propoxyphene, sufentanil, tilidine, tramadol, pharmaceuticallyacceptable derivatives thereof, and mixtures thereof.

Examples of useful non-opioid analgesics belong to, but are not limitedto, the group comprising non-steroidal anti-inflammatory agents, such asaspirin, ibuprofen, diclofenac, naproxen, benoxaprofen, flurbiprofen,fenoprofen, flubufen, ketoprofen, indoprofen, piroprofen, carprofen,oxaprozin, pramoprofen, muroprofen, trioxaprofen, suprofen, aminoprofen,tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac,tolmetin, zomepirac, tiopinac, zidometacin, acemetacin, fentiazac,clidanac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid,niflumic acid, tolfenarnic acid, diflurisal, flufenisal, piroxicam,sudoxicam, and isoxicam.

Examples of other suitable non-opioid analgesics belong to, but are notlimited to, the group comprising analgesics, antipyretics, nonsteroidalanti-inflammatory drugs such as salicylic acid derivatives, includingaspirin, sodium salicylate, choline magnesium trisalicylate, salsalate,diflunisal, salicylsalicylic acid, sulfasalazine, and olsalazin;para-aminophenol derivatives including acetaminophen and phenacetin;indole and indene acetic acids, including indomethacin, sulindac, andetodolac; heteroaryl acetic acids, including tolmetin, diclofenac, andketorolac; anthranilic acids (fenamates), including mefenamic acid andmeclofenamic acid, enolic acids, including oxicams (piroxicam,tenoxicam), and pyrazolidinediones (phenylbutazone, oxyphenthartazone),and alkanones, including nabumetone.

Examples of useful Cox-II inhibitors belong to, but are not limited to,the group comprising rofecoxib and celecoxib.

Examples of useful antimigraine agents belong to, but are not limitedto, the group comprising alpiropride, bromocriptine, dihydroergotamine,dolasetron, ergocornine, ergocorninine, ergocryptine, ergonovine, ergot,ergotamine, flumedroxone acetate, fonazine, ketanserin, lisuride,lomerizine, methylergonovine, methysergide, metoprolol, naratriptan,oxetorone, pizotyline, propranolol, risperidone, rizatriptan,sumatriptan, timolol, trazodone, zolmitriptan, and mixtures thereof.

Examples of useful antiemetic agents belong to, but are not limited to,the group comprising metoclopromide, domperidone, prochlorperazine,promethazine, chlorpromazine, trimethobenzamide, ondansetron,granisetron, hydroxyzine, acetylleucine monoethanolamine, alizapride,azasetron, benzquinamide, bietanautine, bromopride, buclizine,clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron,meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine,thioproperazine, tropisetron, and mixtures thereof.

Examples of useful β-adrenergic blockers belong to, but are not limitedto, the group comprising acebutolol, alprenolol, amosulabol, arotinolol,atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol,bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrinehydrochloride, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol,indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol,moprolol, nadolol, nadoxolol, nebivalol, nifenalol, nipradilol,oxprenolol, penbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sulfinalol, talinolol, tertatolol, tilisolol, timolol,toliprolol, and xibenolol.

Examples of useful anticonvulsants belong to, but are not limited to,the group comprising acetylpheneturide, albutoin, aloxidone,aminoglutethimide, 4-amino-3-hydroxybutyric acid, atrolactamide,beclamide, buramate, calcium bromide, carbamazepine, cinromide,clomethiazole, clonazepam, decimemide, diethadione, dimethadione,doxenitroin, eterobarb, ethadione, ethosuximide, ethotoin, felbamate,fluoresone, gabapentin, 5-hydroxytryptophan, lamotrigine, magnesiumbromide, magnesium sulfate, mephenyloin, mephobarbital, metharbital,methetoin, methsuximide, 5-methyl-5-(3-phenanthryl)-hydantoin,3-methyl-5-phenylhydantoin, narcobarbital, nimetazepam, nitrazepam,oxcarbazepine, paramethadione, phenacemide, phenetharbital, pheneturide,phenobarbital, phensuximide, phenylmethylbarbituric acid, phenyloin,phethenylate sodium, potassium bromide, pregabaline, primidone,progabide, sodium bromide, solanum, strontium bromide, suclofenide,sulthiame, tetrantoin, tiagabine, topiramate, trimethadione, valproicacid, valpromide, vigabatrin, and zonisamide.

Examples of useful antidepressants belong to, but are not limited to,the group comprising binedaline, caroxazone, citalopram, (S)-citalopram,dimethazan, fencamine, indalpine, indeloxazine hydrocholoride, nefopam,nomifensine, oxitriptan, oxypertine, paroxetine, sertraline, thiazesim,trazodone, benmoxine, iproclozide, iproniazid, isocarboxazid, nialamide,octamoxin, phenelzine, cotinine, rolicyprine, rolipram, maprotiline,metralindole, mianserin, mirtazepine, adinazolam, amitriptyline,amitriptylinoxide, amoxapine, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dothiepin, doxepin, fluacizine,imipramine, imipramine N-oxide, iprindole, lofepramine, melitracen,metapramine, nortriptyline, noxiptilin, opipramol, pizotyline,propizepine, protriptyline, quinupramine, tianeptine, trimipramine,adrafinil, benactyzine, bupropion, butacetin, dioxadrol, duloxetine,etoperidone, febarbamate, femoxetine, fenpentadiol, fluoxetine,fluvoxamine, hematoporphyrin, hypericin, levophacetoperane,medifoxamine, milnacipran, minaprine, moclobemide, nefazodone,oxaflozane, piberaline, prolintane, pyrisuccideanol, ritanserin,roxindole, rubidium chloride, sulpiride, tandospirone, thozalinone,tofenacin, toloxatone, tranylcypromine, L-tryptophan, venlafaxine,viloxazine, and zimeldine.

Examples of useful Ca²⁺-channel blockers belong to, but are not limitedto, the group comprising bepridil, clentiazem, diltiazem, fendiline,gallopamil, mibefradil, prenylamine, semotiadil, terodiline, verapamil,amlodipine, aranidipine, barnidipine, benidipine, cilnidipine,efonidipine, elgodipine, felodipine, isradipine, lacidipine,lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, cinnarizine, flunarizine,lidoflazine, lomerizine, bencyclane, etafenone, fantofarone, andperhexyline.

Examples of useful anticancer agents belong to, but are not limited to,the group comprising acivicin, aclarubicin, acodazole hydrochloride,acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantroneacetate, aminoglutethimide, amsacrine, anastrozole, anthramycin,asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat,benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate,bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan,cactinomycin, calusterone, caracemide, carbetimer, carboplatin,carmustine, carubicin hydrochloride, carzelesin, cedefingol,chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicinhydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguaninemesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride,droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin,edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin,enpromate, epipropidine, epirubicin hydrochloride, erbulozole,esorubicin hydrochloride, estramustine, estramustine phosphate sodium,etanidazole, etoposide, etoposide phosphate, etoprine, fadrozolehydrochloride, fazarabine, fenretinide, floxuridine, fludarabinephosphate, fluorouracil, fluorocitabine, fosquidone, fostriecin sodium,gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicinhydrochloride, ifosfamide, ilmofosine, interleukin II (includingrecombinant interleukin II or rIL2), interferon alpha-2a, interferonalpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-I a,interferon gamma-I b, iproplatin, irinotecan hydrochloride, lanreotideacetate, letrozole, leuprolide acetate, liarozole hydrochloride,lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol,maytansine, mechlorethamine hydrochloride, megestrol acetate,melengestrol acetate, melphalan, menogaril, mercaptopurine,methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide,mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper,mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole,nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin,pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan,piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium,porfiromycin, prednimustine, procarbazine hydrochloride, puromycin,puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safingol,safingol hydrochloride, semustine, simtrazene, sparfosate sodium,sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin,streptonigrin, streptozocin, sulofenur, talisomycin, tecogalan sodium,tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxirone,testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin,tirapazamine, toremifene citrate, trestolone acetate, triciribinephosphate, trimetrexate, trimetrexate glucuronate, triptorelin,tubulozole hydrochloride, uracil mustard, uredepa, vapreotide,verteporfin, vinblastine sulfate, vincristine sulfate, vindesine,vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate,vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicinhydrochloride.

Examples of other anti-cancer drugs belong to, but are not limited to,the group comprising 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil;abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor,bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor, interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; 4-ipomeanol; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor, multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor, protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor, urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

Examples of useful therapeutic agents for treating or preventing UIbelong to, but are not limited to, the group comprising propantheline,imipramine, hyoscyamine, oxybutynin, and dicyclomine.

Examples of useful therapeutic agents for treating or preventing anulcer belong to, but are not limited to, the group comprising antacidssuch as aluminum hydroxide, magnesium hydroxide, sodium bicarbonate, andcalcium bicarbonate; sucraflate; bismuth compounds such as bismuthsubsalicylate and bismuth subcitrate; H₂ antagonists such as cimetidine,ranitidine, famotidine, and nizatidine; H⁺, K⁺-ATPase inhibitors such asomeprazole, iansoprazole, and lansoprazole; carbenoxolone; misprostol;and antibiotics such as tetracycline, metronidazole, timidazole,clarithromycin, and amoxicillin.

Examples of useful therapeutic agents for treating or preventing IBDbelong to, but are not limited to, the group comprising anticholinergicdrugs; diphenoxylate; loperamide; deodorized opium tincture; codeine;broad-spectrum antibiotics such as metronidazole; sulfasalazine;olsalazie; mesalamine; prednisone; azathioprine; mercaptopurine; andmethotrexate.

Examples of useful therapeutic agents for treating or preventing IBSinclude belong to, but are not limited to, the group comprisingpropantheline; muscarine receptor antogonists such as pirenzapine,methoctramine, ipratropium, tiotropium, scopolamine, methscopolamine,homatropine, homatropine methylbromide, and methantheline; andantidiarrheal drugs such as diphenoxylate and loperamide.

Examples of useful therapeutic agents for treating or preventing anaddictive disorder belong to, but are not limited to, the groupcomprising methadone, desipramine, amantadine, fluoxetine,buprenorphine, an opiate agonist, 3-phenoxypyridine, levomethadylacetate hydrochloride, and serotonin antagonists.

Examples of useful therapeutic agents for treating or preventingParkinson's disease and parkinsonism belong to, but are not limited to,the group comprising carbidopa/levodopa, pergolide, bromocriptine,ropinirole, pramipexole, entacapone, tolcapone, selegiline, amantadine,diphenhydramine, apomorphine, ethopropazine, benztropine mesylate,lergotril, biperiden, lisuride, metixen, chlorphenoxamine, orphenadrine,cycrimine, procyclidine, dexetimide, trihexyphenidyl, andtrihexyphenidyl hydrochloride.

Examples of useful therapeutic agents for treating or preventing anxietybelong to, but are not limited to, the group comprising benzodiazepines,such as alprazolam, brotizolam, chlordiazepoxide, clobazam, clonazepam,clorazepate, demoxepam, diazepam, estazolam, flumazenil, flurazepam,halazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam,prazepam, quazepam, temazepam, and triazolam; non-benzodiazepine agents,such as buspirone, gepirone, ipsaprione, tiospirone, zolpicone,zolpidem, and zaleplon; tranquilizers, such as barbituates, e.g.,amobarbital, aprobarbital, butabarbital, butalbital, mephobarbital,methohexital, pentobarbital, phenobarbital, secobarbital, andthiopental; and propanediol carbamates, such as meprobamate andtybamate.

Examples of useful therapeutic agents for treating or preventingepilepsy belong to, but are not limited to, the group comprisingcarbamazepine, ethosuximide, gabapentin, lamotrignine, phenobarbital,phenyloin, primidone, valproic acid, trimethadione, bemzodiaepines,gabapentin, lamotrigine, γ-vinyl GABA, acetazolamide, and felbamate.

Examples of useful therapeutic agents for treating or preventing strokebelong to, but are not limited to, the group comprising anticoagulantssuch as heparin, agents that break up clots such as streptokinase ortissue plasminogen activator, agents that reduce swelling such asmannitol or corticosteroids, and acetylsalicylic acid.

Examples of useful therapeutic agents for treating or preventing aseizure belong to, but are not limited to, the group comprisingcarbamazepine, ethosuximide, gabapentin, lamotrignine, phenobarbital,phenyloin, primidone, valproic acid, trimethadione, bemzodiaepines,gabapentin, lamotrigine, γ-vinyl GABA, acetazolamide, and felbamate.

Examples of useful therapeutic agents for treating or preventing apruritic condition belong to, but are not limited to, the groupcomprising naltrexone; nalmefene; danazol; tricyclics such asamitriptyline, imipramine, and doxepin; antidepressants such as thosegiven below, menthol; camphor, phenol; pramoxine; capsaicin; tar;steroids; and antihistamines.

Examples of useful therapeutic agents for treating or preventingpsychosis belong to, but are not limited to, the group comprisingphenothiazines such as chlorpromazine hydrochloride, mesoridazinebesylate, and thoridazine hydrochloride; thioxanthenes such aschloroprothixene and thiothixene hydrochloride; clozapine; risperidone;olanzapine; quetiapine; quetiapine fumarate; haloperidol; haloperidoldecanoate; loxapine succinate; molindone hydrochloride; pimozide; andziprasidone.

Examples of useful therapeutic agents for treating or preventingHuntington's chorea belong to, but are not limited to, the groupcomprising haloperidol and pimozide.

Examples of useful therapeutic agents for treating or preventing ALSbelong to, but are not limited to, the group comprising baclofen,neurotrophic factors, riluzole, tizanidine, benzodiazepines such asclonazepan and dantrolene.

Examples of useful therapeutic agents for treating or preventingcognitive disorders belong to, but are not limited to, the groupcomprising agents for treating or preventing dementia such as tacrine;donepezil; ibuprofen; antipsychotic drugs such as thioridazine andhaloperidol; and antidepressant drugs such as those given above.

Examples of useful therapeutic agents for treating or preventing amigraine belong to, but are not limited to, the group comprisingsumatriptan; methysergide; ergotamine; caffeine; and beta-blockers suchas propranolol, verapamil, and divaiproex.

Examples of useful therapeutic agents for treating or preventingvomiting belong to, but are not limited to, the group comprising 5-HT₃receptor antagonists such as ondansetron, dolasetron, granisetron, andtropisetron; dopamine receptor antagonists such as prochlorperazine,thiethylperazine, chlorpromazin, metoclopramide, and domperidone;glucocorticoids such as dexamethasone; and benzodiazepines such aslorazepam and alprazolam.

Examples of useful therapeutic agents for treating or preventingdyskinesia belong to, but are not limited to, the group comprisingreserpine and tetrabenazine.

Examples of useful therapeutic agents for treating or preventingdepression belong to, but are not limited to, the group comprisingtricyclic antidepressants such as amitryptyline, amoxapine, bupropion,clomipramine, desipramine, doxepin, imipramine, maprotilinr, nefazadone,nortriptyline, protriptyline, trazodone, trimipramine, and venlaflaxine;selective serotonin reuptake inhibitors such as citalopram,(S)-citalopram, fluoxetine, fluvoxamine, paroxetine, and setraline;monoamine oxidase inhibitors such as isocarboxazid, pargyline,phenelzine, and tranylcypromine; and psychostimulants such asdextroamphetamine and methylphenidate.

Examples of other useful pharmaceutical compounds can belong to, but arenot limited to, the group of corticosteroids comprisingmineralocorticosteroids such as cortisol, desoxycorticosterone andfluorohydrocortisone, lucocorticosteroids such as beclomethasone,betamethasone, cortisone, dexamethasone, fluocinolone, fluocinonide,fluocortolone, fluorometholone, fluprednisolone, flurandrenolide,halcinonide, hydrocortisone, medrysone, methylprednisolone,paramethasone, prednisolone, prednisone and triamcinolone (acetonide),androgens comprising androgenic steroids used in therapy such asdanazole, fluoxymesterone, mesterolone, methyltestosterone, andtestosterone and salts thereof, anabolic steroids used in therapy suchas calusterone, nandrolone and salts thereof, dromostanolone,oxandrolone, ethylestrenol, oxymetholone, methandriol, stanozolol,methandrostenolone and testolactone, anti-androgens such as cyproteroneacetate, estrogens comprising estrogenic steroids used in therapy suchas diethylstilbestrol, estradiol, estriol, ethinylestradiol, mestranolor quinestrol, anti-estrogens such as chlorotrianisene, clomiphene,ethamoxytriphetol, nafoxidine and tamoxifen, progestins such asallylestrenol, desogestrel, dimethisterone, dydrogesterone,ethinylestrenol, ethisterone, ethynadiol diacetate, etynodiol,hydroxyprogesterone, levonorgestrel, lynestrenol, medroxyprogesterone,megestrol acetate, norethindrone, norethisterone, norethynodrel,norgestrel, and progesterone.

The pharmaceutical active compound can be also a peptide or protein,e.g. an enzyme such as lysozyme. The terms “peptide”, “polypeptide” or“protein” may be used interchangeably. The methods to determine whethera peptide or a protein is suitable for loading, i.e. is water-solubleand/or carries a net charge at a given pH-value is known to one of skillin the art, e.g. described in F. Lottspeich/Z. Zorbas [Lottspeich, F.;Zorbas, H. (Hrsg.) Bioanalytik Spektrum Akademischer Verlag: Heidelberg,1998]. Relatively small peptides may be referred to by the number ofamino acids (e.g. di-, tri-, tetrapeptides). A peptide having arelatively small number of amide bonds may also be called anoligopeptide (up to 50 amino acids), whereas a peptide with a relativelyhigh number (more than 50 amino acids) may be called a polypeptide orprotein. In addition to being a polymer of amino acid residues, certainproteins may further be characterized by the so called quaternarystructure, a conglomerate of a number of polypeptides that are notnecessarily chemically linked by amide bonds but are bonded by forcesgenerally known to the skilled person, such as electrostatic forces andvan-der-Waals forces. The term peptides, proteins or mixtures thereof asused herein is to include all above mentioned possibilities. Usually,the protein and/or peptide are selected on the basis of its biologicalactivity.

Other examples of peptides or proteins or entities comprising peptidesor proteins, which may advantageously be loaded onto and/or into thesilk particles, preferably spider silk particles, according to theinvention belong to, but are not limited to, the group comprisingimmunogenic peptides or immunogenic proteins which comprise thefollowing:

Examples of useful toxins belong to, but are not limited to, the groupcomprising diphtheria toxin and tetanus toxin.

Examples of useful viral surface antigens or parts of viruses belong to,but are not limited to, the group comprising adenoviruses, Epstein-BarrVirus, Hepatitis A Virus, Hepatitis B Virus, Herpes viruses, HIV-1,HIV-2, HTLV-III, Influenzaviruses, Japanese encephalitis virus, Measlesvirus, Papilloma viruses, Paramyxoviruses, Polio Virus, Rabies, Virus,Rubella Virus, Vaccinia (Smallpox) viruses and Yellow Fever Virus.

Examples of useful proteins belong to, but are not limited to, the groupof bacterial surface antigens or parts of bacteria such as Bordetellapertussis, Helicobacter pylori, Clostridium tetani, Corynebacteriumdiphtheria, Escherichia coli, Haemophilus influenza, Klebsiella species,Legionella pneumophila, Mycobacterium bovis, Mycobacterium leprae,Mycrobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Proteus species, Pseudomonas aeruginosa, Salmonellaspecies, Shigella species, Staphylococcus aureus, Streptococcuspyogenes, Vibrio cholera or Yersinia pestis.

Examples of useful proteins belong to, but are not limited to, the groupof surface antigens of parasites causing disease or portions ofparasites such as Plasmodium vivax (malaria), Plasmodium falciparum(malaria), Plasmodium ovale (malaria), Plasmodium malariae (malaria),Leishmania tropica (leishmaniasis), Leishmania donovani),leishmaniasis), Leishmania branziliensis (leishmaniasis), Trypanosomarhodescense (sleeping sickness), Trypanosoma gambiense (sleepingsickness), Trypanosoma cruzi (Chagas' disease), Schistosoma mansoni(schistosomiasis), Schistosomoma haematobium (schistomiasis),Schistosoma japonicum (shichtomiasis), Trichinella spiralis(trichinosis), Stronglyloides duodenale (hookworm), Ancyclostomaduodenale (hookworm), Necator americanus (hookworm), Wucheria bancrofti(filariasis), Brugia malaya (filariasis), Loa loa (filariasis),Dipetalonema perstaris (filariasis), Dracuncula medinensis (filariasis),or Onchocerca volvulus (filariasis).

Examples of useful proteins belong to, but are not limited to, the groupof antitoxins such as Botulinum antitoxin, diphtheria antitoxin, gasgangrene antitoxin and tetanus antitoxin.

Examples of useful proteins belong to, but are not limited to, the groupof antigens which elicit an immune response against foot and mouthdisease. Examples of useful proteins belong to, but are not limited to,the group of hormones and growth factors such as follicle stimulatinghormone, prolactin, angiogenin, epidermal growth factor, calcitonin,erythropoietin, thyrotropic releasing hormone, insulin, growth hormones,insulin-like growth factors 1 and 2, skeletal growth factor, humanchorionic gonadotropin, luteinizing hormone, nerve growth factor,adrenocorticotropic hormone (ACTH), luteinizing hormone releasinghormone (LHRH), parathyroid hormone (PTH), thyrotropin releasing hormone(TRH), vasopressin, cholecystokinin, and corticotropin releasinghormone; cytokines, such as interferons, interleukins, colonystimulating factors, and tumor necrosis factors: fibrinolytic enzymes,such as urokinase, kidney plasminogen activator; and clotting factors,such as Protein C, Factor VIII, Factor IX, Factor VII or AntithrombinIII.

Examples of other proteins or peptides belong to, but are not limitedto, the group of albumin, atrial natriuretic factor, renin, superoxidedismutase, alpha 1-antitrypsin, lung surfactant proteins, bacitracin,bestatin, cydosporine, delta sleep-inducing peptide (DSIP), endorphins,glucagon, gramicidin, melanocyte inhibiting factors, neurotensin,oxytocin, somostatin, terprotide, serum thymide factor, thymosin, DDAVP,dermorphin, Met-enkephalin, peptidoglycan, satietin, thymopentin, fibrindegradation product, des-enkephalin-alpha-endorphin, gonadotropinreleasing hormone, leuprolide, alpha-MSH or metkephamid.

Preferred useful therapeutic agents are selected from the groupconsisting of tetracaine, procaine, papaverine, ephedrine, propanolol,and ecthacridine lactate.

As used herein, the terms “cosmetic substances” and “cosmetic compounds”may be used interchangeably and designate substances intended mainly forexternal use on the human body or in the oral cavity for cleaning andpersonal hygiene to alter the appearance or body odor or to conveyscent. In particular, it is meant that cosmetic substances are moleculeswhich show a certain predictable effect. Such effect molecules can befor example proteinaceous molecules such as enzymes or non-proteinaceousmolecules such as dyes, pigments, photoprotective agents, vitamins,provitamins, antioxidants, conditioners or compounds comprising metalions.

Among the proteinaceous molecules enzymes and antibodies are preferred.Examples for useful belong to, but are not limited to, the groupcomprising oxidases, peroxidases, proteases, glucanases, mutanase,tyrosinases, laccases, metal-binding enzymes, lactoperoxidase, lysozyme,aminoglycosidase, glucose oxidase, super oxide dismutase, photolyase, T4endonuclease, catalase, thioredoxin or thioredoxin-reductase.

Also preferable are proteinaceous substances which do not possess anenzymatic function. Examples for non-enzymatic proteinaceous moleculesbelong to, but are not limited to, the group comprising antimicrobialpeptides, hydrophobins, collagen, proteins binding carotenoid, proteinsbinding heavy metals, proteins binding odorants, proteins bindingcellulose, proteins binding starch or proteins binding keratin.

Examples of useful proteinaceous molecules belong to, but are notlimited to, the group comprising protein hydrolysates of plant or animalsources. For example, the protein hydrolysate can be of marine origin.

The cosmetic compound can further be a UV-protective filter. These areby definition organic substances which can absorb specific wavelengthsin the range of UV-wavelengths. The absorbed energy can then emitted inform of longer wave radiation, e.g. heat.

Examples of suitable water-soluble UV-protective filters belong to, butare not limited to, the group comprising to,2-phenyl-benzimidazole-5-sulfonic acid and the alkali metal, alkalineearth metal, ammonium, alkylammonium, alkanolammonium and glucammoniumsalts thereof, sulfonic acid derivatives of benzophones such as2-hydroxxy-4-methoxybenzophene-5-sulfonic acid and its salts, sulfonicacid derivatives of 3-benzylidenecamphor such as4-(2-oxo-3-bornylidene-methyl)benzenesulfonic acid and2-methyl-5-(2-oxo-3-bornylidene)-sulfonic acid and salts thereof, estersof cinnamic acid such as 2-ethylhexyl 4-methoxycinnamate, isopentyl4-methoxycinnamate or 2-ethylhexyl 2-cyano-3-phenylcinnamate(octocrylene), derivatives of benzophene such as2-hydroxy-4-methoxybenzophne,2-hydroxy-4-methoxy-4′-methyl-benzophenone,2,2′-dihydroxy-4-methoxybenzophenone or propane-1,3-diones such as1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione.

The cosmetic compound may also comprise a secondary protective agent ofthe antioxidant type which interrupts the photochemical reaction chaintriggered by UV radiation when penetrating into the skin. Typicalexamples belong to, but are not limited to, the group comprising superoxide dismutase, catalase, tocopherols (vitamin E), coenzyme Q10,ubiquinanes, quiniones and ascorbic acid (vitamin C).

The cosmetic, compound can also be a vitamin, a provitamin or precursorsthereof. Examples belong to, but are not limited to, the groupcomprising β-carotene (provitamin of vitamin A), ascorbic acid (vitaminC), tocopherols, the vitamins, provitamins or precursors of the vitaminB group or derivatives thereof such as vitamin B₁ (thiamine), vitamin B₂(riboflavin) or the stereoisomer lyxoflavin, vitamin B₃ (nicotinic acidor nicotinamid), vitamin B5 (panthothenic acid and panthenol) andderivatives thereof such as esters, ethers and cationically derivatizedpanthenol, derivatives of 2-furanone such asdihydro-3-hydroxy-4,4-dimethyl-2(3H)-furanone (pantolactone),4-hydroxymethyl-γ-butyrolactone, 3,3-dimethyl-2-hydroxy-γ-butyrolactoneand 2,5-dihydro-5-methoxy-2-furanone and stereoisomers thereof, vitaminB₆ such as derivatives of 5-hydroxymethyl-2-methylpyridin-3-ol (alsoknown as pyridoxine, pyridoasamine or pyridoxal) and vitamin B₇(biotin).

Examples of useful cosmetic compounds belong to, but are not limited to,the group of antioxidants, comprising amino acids such as tyrosine andcysteine and derivatives thereof, and tannins.

Examples of useful cosmetic compounds belong to, but are not limited to,the group of peroxide decomposers comprisingpyridine-2-thiol-3-carboxylic acid, 2-methoxypyrimidinolcarboxylicacids, and 2-dimethylaminopyridinecarboxylic acids.

The cosmetic compound can also comprise dyes such as food dyes,semi-permanent dyes, reactive or oxidation dyes. Examples of useful dyesare for example described in Rowe Colour Index, 3^(rd) edition, Societyof Dyers and Colourists, Bradford, England, 1971.

As used herein, the terms “agricultural substance”, “agriculturalcompound” and “agricultural active ingredient” can be usedinterchangeably and means chemicals (including veterinary medicines)used in the production of primary produce (farmed plants or animals).They are also used by home gardeners, and for the health of domesticanimals such as cats and dogs. Agricultural compounds can be any naturalor synthetic and include substances such as veterinary medicines,fertilisers and pesticides.

The agricultural active ingredient may be a pesticide, selected from thegroup such as insecticides, nematocides, fungicides and herbicides; andpossibly molluscicides and rodenticides.

Examples of useful agricultural active ingredients belong to, but arenot limited to, the group comprising organophosphates, carbamates,benzimidazoles dicarboxamides, bipyridols, pyrethroids and chlorinatedhydrocarbons.

Examples of useful organophosphates belong to, but are not limited to,the group comprising azinphos methyl, dimethoate, ethyl parathion,trichlorfon, dibrom, dimecron, mevinphos, and monocrotophos.

Examples of useful carbamates belong to, but are not limited to, thegroup comprising methomyl, oxamyl, aldicarb, carbofuran, fenoxycarb,carbaryl, ethionocarb, and fenobucarb.

Examples of useful benzimidazole belong to, but are not limited to, thegroup comprising as benomyl, carbendaz or thiophanate-methyl.

Examples of useful dicarboxamides belong to, but are not limited to, thegroup comprising vinclozolin, iprodione, procymidone or captan.

Examples of useful bipyridols belong to, but are not limited to, thegroup comprising paraquat and diquat.

The agricultural compound may also be a pyrethroid. Examples of usefulpyrethroids belong to, but are not limited to, the group comprisingcypermethrin or a chlorinated hydrocarbon such as DDT, dicofol,heptachlor, endosulfan, chlordane, aldrin, dieldrin, endrin, mirex, andpentachlorphenol.

The agricultural compound may also be a synthetic organic fertilizersuch as urea.

The term “chemoattractant” means organic or inorganic substancespossessing chemotacis inducer effect in motile cells. Effects ofchemoattractants are elicited via described or hypothetic chemotaxisreceptors, the chemoattractant moiety of a ligand is target cellspecific and concentration dependent. Most frequently investigatedchemoattractants are formyl peptides and chemokines.

Chemokines are a family of small cytokines, or proteins secreted bycells. Proteins are classified as chemokines according to sharedstructural characteristics such as small size (8-10 kD in size); and thepresence of four cysteine residues in conserved locations that are keyto forming their 3-dimensional shape.

Examples of useful chemokines belong to, but are not limited to, thegroup of the chemokine family including CC-chemokines (or β-chemokines)such as I-309, MCP-1, MEP-1α, MIP-1β, RANTES, C10 (MRP-2), MARC (MCP-3),MCP-2, MRP-2, Eotaxin, MCP-5, MCP-4, HCC-1, Leukotactin-1, LEC (NCC-4),TARC, PARC, ELC, LARC, SLC, MDC, MPIF-1, Eotaxin-2, TECK, Eotaxin-3,CTACK or MEC, CXC chemokines (or α-chemokines) such as Gro-α, Gro-β,Gro-γ, PF-4, ENA-78, GCP-2, NAP-2, IL-8, MIG, IP-10, I-TAC, SDF-1,BCA-1, BRAK, Lungkine, SRPDOC or VCC-1, C-chemokines such aslymphoctactin α and lymphotactin β, and CX₃C-chemokines such asfractalkine.

As used herein, “chemorepellents” are substances expressing adversemigratory effect. These are typically compounds capable of repelling (orchemorepelling) a eukaryotic cell with migratory capacity, i.e. a cellthat can move away from a repellant stimulus.

Examples of useful chemorepellents belong to, but are not limited to,the group comprising amino acids and chemokines such as IL-8 or SDF-1.

The terms “anti-fungal substance” or “fungizide” can be usedinterchangeably. By definition fungicides are chemical compounds whichinhibit fungi or fungal spores. It is meant that fungicides aresubstances used both in agriculture and to fight fungal infectionsinanimals (antifungal drug). Chemicals used to control oomycetes, whichare not fungi, are also referred to as fungicides since oomycetes usethe same mechanisms as fungi to infect plants.

Examples of used antifungal drugs belong to, but are not limited to, thegroup comprising polyene antifungals such as natamycin, rimocidin,filipin, nystatin, amphotericin B or candicin, imidazoles such asmiconazole, ketoconazole, clotrimazole, econazole, bifonazole,butaoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole,sulconazole, tioconazole or gresofluin, thiazoles such as fluconazole,itraconazole, isavulconazole, ravuconazole, posaconazole, terconazole orvoriconazole, thiazoles such as abafungin, allylamines such asterinafine, amorolfine, naftifine or bunafine, echinocandins such asanidulafungin, caspofungin or micafungin.

Examples of other anti-fungal drugs belong to, but are not limited to,the group comprising ciclopirox olamine, tolnaftate, flucytosine,griseofluvin or haloprogin.

As used herein, a “nutrient” is a chemical that an organism needs tolive and grow or a substance used in an organism's metabolism which mustbe taken in from its environment. Organic nutrients includecarbohydrates, fats, proteins (amino acids), and vitamins. Inorganicnutrients are dietary minerals, water, and oxygen. Preferred nutrientsare macronutrients such as carbohydrates, amino acids or proteins andmicronutrients such as vitamins.

Examples of useful carnohydrates belong to, but are not limited to, thegroup od monosaccharides such as, glyceraldehyde, erythrose, threose,ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose,gulose, idose, galactose, talose, dihydroxacetone, erythrulose,ribulose, xylulose, psicose, fructose, sorbose, tagatose orstereoisomers thereof, amino sugars such as galactosamine, glucosamine,sialic acid, N-acetylglucosamine, sulfosugars such as sulfoquinovose,disaccharides such as sucrose, lactulose, lactose, maltose, trehalose ormaltobiose, or oligosacharides such as Fructooligosaccharides (FOS),Galactooligosaccharides (GOS) or Mannan-oligosaccharides (MOS).

The terms “dietary supplement”, “food supplement” or “nutritionalsupplement” as used herein, refer to a preparation intendended nutrientssuch as vitamins, minerals, fiber, fatty acids or amino acids, that aremissing or are not consumed in sufficient quantitiy in a person's diet.Depending on the country dietary supplements are either definded asfoods or as drugs.

Examples of other dietary supplements belong to, but are not limited to,the group comprising steroids such as dehydroepiandrosterone (DHEA),pregnenolone, or derivatives thereof, hormones such as melatonin, andother substances such as hydrrazine sulfate, caffeine(1,3,7-trimethylxanthine), catechins, soy isoflavones, glucosamine,coenzyme-Q10, ephedrine-type alkaloids such as ephedra or ephedrine,synephrine, norephedrine, or pseudodoephedrine.

The term “dye” as used herein refers to a coloured substance havingaffinity to a substrate to which it is being applied. Dyes are generallyapplied in aqueous solution. In contrast, pigments are typicallyinsoluble and possess no affinity to the substrate. Both dyes andpigments appear to be coloured because of their ability to absorbspecific wavelength of light. The dye can be a naturally occurring orsynthetic organic dye or a food dye.

Examples of useful dyes belong to, but are not limited to, the group ofa acridine dyes such as acridine orange or acridine yellow,anthrachinone dyes such as Alizarin, Anthrapurpurin, Carminic acid,Disperse Red 11, Disperse Red 9, Indathrene blue RS, Morindone, Oil blue35, Oil blue A, Quinizarine Green SS, Solven violet 13 or Vat Yellow 4,diarylmethane dyes such as the diarylmethane dye auramine O ortriarylmethanes such as Aluminon, Aniline Blue WS, Aurin, Brilliant BlueFCF, Brilliant Green, Bromocresol green, Bromocresol purple, Bromophenolblue, Bromothymol blue, Bromosulphtalein, Chlorophenol red, Chromoxanecyanin R, Coomassie, Cresol red, Crystal violet lactone, Ethyl Green,Fast Green FCF, Fluoran, Fuchsin, Fuchsin acid, Green S, Light Green SFyellowish, Malachite green, Methyl violet, Methyl blue,Methylrosaniline, New fuchsine, pararosaniline, Patent Blue V, Phenolred, Phenolphtalein, Rose bengal, Thymolphtalein, Victoria blue BO,Xylene cyanol or Xylenol orange, azo dyes such as Alizarine Yellow R,Allura Red AC, Amaranth, Amido black 10 B, Aniline Yellow, Azo rubine,Biebrich scarlet, Bismarck brown Y, Black 7984, Brilliant black BN,Brown FK, Brown HT, Chrysoine resorcinol, Citrus red 2, Congo red, D&CRed 33, Disperse Orange 1, Eriochrome Black T, Fast Yellow AB,Hydroxynaphtol blue, Janus Green B, Lithol Rubine BK, Lithiol Rubine BK,Methyl orange, Methyl Red, Methyl yellow, Mordant Red 19, Oil Red O, OilYellow DE, Orange B, Orange G, Orange GGN, Para Red, Ponceau 2R, Ponceau4R, Ponceau 6R, Ponceau S, Prontosil, Red 2G, Scarlet GN, Solvent Red164, Solvent Red 26, Solvent Yellow 124, Sudan Black B, Sudan I, SudanII, Sudan III, Sudan IV, Sudan Red 7B, Sudan Red G, Sudan Yellow 3G,Sudan Yellow FCF, Tartrazine, Tropaeolin OO, Tropaeolin OOO or Trypanblue, cyanin dyes (or phtalocyanines) such as Alcian blue, Luxol fastblue, Direct blue 86, Direct blue 199, Phtalocyanine blue BN orPhtalocyanine green GN, azin dyes such as Neutral Red or Safranin, Nitrodyes such as picric acid and martius yellow, indolphenol dyes such asdichlorophenolindophenol, oxazin dyes such as nile blue, nile red,gallocyanin, gallamin blue or celestin blue, thiazin dyes such asmethylene blue or new methylene blue or toluidine blue O, xanthene dyesor derivatives thereof including fluorescein, eosins such as Eosin Y andEosin B and rhodamines such as Rhodamine B, Rhodamine 6G, Rhodamine 123,pyronin dyes such as Pyronin B and Pyronin Y, tetramethylrhodamine(TAMRA) and its isothiocyanate derivative (TRITC), sulforhodamine 101and its sulfonyl chloride form Texas Red and Rhodamine Red or newerfluorophores such as Alexa dyes, e.g. Alexa 546, Alexa 555, Alexa 633,or Dylight dyes, e.g. DyLight 549, DyLight 633, or a mixture thereof.

The terms “fragrance”, “odorant” “aroma”, “aroma compound” or “flavour”can be used interchangeably and refer to a chemical compound that has asmell or odor. Typically, a chemical compound possess a smell or odorwhen the compound is essentially volatile, so it can be transported tothe olfactory in the upper part of the nose in sufficiently highconcentrations to be able to interact with one or more of the olfactoryreceptors.

Examples of useful aroma compounds belong to, but are not limited to,the group of esters such as methyl formate, methyl acetate, methylbutyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentylbutyrate, pentyl pentanoate, octyl acetate, fructone, hexy acetate orethyl methylphenylglycidate, terpenes such as myrcene, geraniol, nerol,citral, citronellal, citronellol, linalool or nerolidol, cyclic terpenessuch as limonene, camphor, terpineol, alpha-ionone, terpineol, thujone,aromatic compounds such as benzaldehyde, eugenol, cinnamaldehyde, ethylmaltol, vanillin, anisole, anethole, estragole or thymol, amines such astrimethylamine, putrescine, cadaverine, pyridine, indole or skatole,alcohols such as furaneol, 1-hexanol, cis-3-hexen-1-ol or menthol,aldehydes such as acetaldehyde, hexanal, cis-3-hexenal, furfural,ketones such as dihydrojasmone, oct-1-en-3-one, 2-acetyl-1-pyrroline,6-acetyl-2,3,4,5-tetrahydropyridine, lactones such as gamma-decalactone,gamma-nonalactone, delta-octalocatone, jasmine lactone, massoia lactone,wine lactone or sotolon, thiols such as ethanethiols, nerolin,tetrahydrothiophene, 2,4,6-trichloranisole or substituted pyrazines, andmixtures thereof.

The compound can be also any other agent such as a hemostatic agent suchas sulmarin, carbazochrome, etamsylate, calcium dobesilate, esculamine,oxamarin, ornipressin, desmopressin, felypressin, octreotide, poliglusamor aprotinin.

Examples of useful other hemostatic compounds belong to, but are notlimited to, the group comprising different, suitable hydrates such aspotassium aluminum sulfate, aluminum sulfate, aluminum iron sulfate,aluminum ammonium sulfate, iron chloride, aluminum chloride, sodiumchloride, zinc chloride, zinc phenol sulfate, tannic acids andadrenalin.

The other agent can also be a growth stimulating agent. The terms“growth stimulating agent”, “growth factor” and “growth horomone” may beused interchangeably and refer to substances capable of stimulatingcellular growth, proliferation and cellular differentiation. Typicallythese agents a proteins or steroids hormones. Growth factors areimportant for regulating a variety of cellular processes. Growth factorstypically act as signaling molecules between cells. Examples arecytokines and hormones that bind to specific receptors on the surface oftheir target cells.

Examples of suitable growth stimulating belong to, but are not limitedto, the group comprising bone morphogenetic proteins (BMPs), epidermalgrowth factors (EGF), erythropoietin (EPO), fibroblast growth factor(FG), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage stimulating factor (GM-CSF), growthdifferentiation factor-9 (GDF9), hepatocyte growth factor (HGF),hepatoma derived growth factor (HDGF), insulin-like growth factor(HDGF), insulin-like growth factor (IGF), myostatin (GDF8), nerve-growthfactor (NGF), platelet-dervived growth factor (PDGF), transforminggrowth factor alpha (TGF-α), transforming growth factor beta (TGF-β),and vascular ecdothelial growth factor (VEGF).

The other agent can be also an anti-fouling agent. The term“anti-fouling agent” as used herein refer to an agent that inhibits thegrowth of barnacles and other marine organisms on a ship's bottom (anantifouling paint or other coating).

Examples of useful anti-fouling agents belong to, but are not limitedto, the group comprising irgarol 1051, copper- or zinc pyrithione,diuron and isothioazolinons such as Sea-nine 211.

The terms “proinflammatory agent” or “inflammatory agent” herein referto any substance produced in an animal that is a direct or indirectmediator of inflammation, or is directly or indirectly involved inproduction of a mediator of inflammation. A variety of proinflammatorysubstances are known to those skilled in the art.

Examples of useful proinflammatory substances include belong to, but arenot limited to, the group comprising eicosanoids such as prostaglandins,e.g., PGE2 and leukotrienes e.g., LTB4, enzymes such as phospholipases,inducible nitric oxide synthase (iNOS), COX-1 and COX-2 and cytokinessuch as interleukins (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-8, IL-10, IL-12 and IL-18), members of the tumor necrosis factorfamily, e.g. TNF-α, TNF-β and lymphotoxin β, interferons, e.g., IFN-βand IFN-γ, granulocyte/macrophage colony-stimulating factor (GM-CSF),transforming growth factors such as TGF-β1, TGF-β2 and TGF-β3, leukemiainhibitory factor (LIF), ciliary neurotrophic factor (CNTF), migrationinhibitory factor (MIF), monocyte chemoattractant protein (MCP-1),macrophage inflammatory proteins (e.g., MIP-1α, MIP-1β and MIP-2), andRANTES.

Examples of other suitable substances having pro-inflammatory activitybelong to, but are not limited to, the group comprising bacterialcomponents such as lipopolysaccaride (LPS), teichoic and lipoteichoicacids, peptidoglycans, bacterials DNA such as fragments containingCpG-motifs, bacterial proteins such as entero- and exotoxins orhemolysins such as pneumoslysins, and yeast cell wall component such aszymosan.

In further specific embodiments, step ii) of the method is carried outat temperatures of between 4° C. and 40° C., preferably of between 10°C. and 30° C. and more preferably of between 20° C. and 25° C., e.g. 4°C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C.,14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C.,23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C.,32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40°C.

In further specific embodiments, step ii) of the method is carried outat a pH of between 1 and 9, preferably of between 4 and 9 and mostpreferably of between 6 and 8, e.g. pH 1, 2, 3, 4, 5, 6, 7, 8, or 9.

All features and characteristics according to the first aspect alsoapply to further aspects of the present invention as are described asfollows.

In a second aspect, the present invention relates to silk particles,preferably spider silk particles, comprising at least one silkpolypeptide, preferably spider silk polypeptide, comprising at least twoidentical repetitive units loaded with at least one compound, which ispreferably water-soluble and/or has a molecular weight of between about50 Da and about 20 kDa.

A compound which is well-suited for efficient loading of the silkparticles, e.g. spider silk particles, is sufficiently small in size. Ina preferred embodiment of the invention, the compound has a molecularweight of 50 Da or about 50 Da to 20 kDa or about 20 kDa; or 50 Da orabout 50 Da to 10 kDa or about 10 kDa, preferably 50 Da or about 50 Dato 6 kDa or about 6 kDa, more preferably 50 Da or about 50 Da to 4 kDaor about 4 kDa and most preferably 50 Da or about 50 Da to 1 kDa orabout 1 kDa, e.g. 50 Da, 100 Da, 150 Da, 200 Da, 250 Da, 300 Da, 350 Da,400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650 Da, 700 Da, 750 Da, 800 Da,850 Da, 900 Da, 950 Da, 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa,4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa, 8 kDa,8.5 kDa, 9 kDa, 9.5 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa,16 kDa, 17 kDa, 18 kDa, 19 kDa, or 20 kDa.

Further, a compound which is well-suited for efficient loading of thesilk particles, e.g. spider silk particles, is preferably water-soluble.

Furthermore, a preferred compound according to the invention may be anycompound, which is a small and water-soluble compound, preferably havinga molecular weight of between about 50 Da and 20 kDa, more preferably 50Da to 10 kDa or 50 Da to 6 kDa and most preferably 50 Da to 4 kDa or 50Da to 1 kDa (see above).

As mentioned above, the compound is able to permeate into the silkmatrix, preferably spider silk matrix. Preferably, at least 40%, morepreferably 50%, 60%, 70%, 80%, 90%, or 95% of the loaded compound islocated within the matrix of the silk particles, preferably spider silkparticles, e.g. at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, or 95%.

In preferred embodiments of the invention, the median size of theparticles is 0.1 μm to 500 μm, preferably 0.1 μm to 100 μm, morepreferably 0.2 μm to 20 μm, even more preferably 0.2 μm to 1 μm and mostpreferably 0.25 μm to 0.7 μm, e.g. 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4,0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500

In further specific embodiments, the silk polypeptide, preferably spidersilk polypeptide, comprises, essentially consists of, or consists of atleast two identical repetitive units each comprising at least one,preferably one, consensus sequence selected from the group consistingof:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from the group consisting of        A, S, G, Y, P and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from the group consisting of Y, P, R, S,        A, T, N and Q; and    -   iii) A_(x), wherein x is an integer from 5 to 10.

It is also preferred that the silk polypeptide comprises, essentiallyconsists of, or consists of at least two identical repetitive units eachcomprising at least one, preferably one, amino acid sequence selectedfrom the group consisting of: GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG(SEQ ID NO: 19). The GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO:19) (peptide) motifs have been selected from Resilin (WO 08/155304).

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of between 2 to 80 repetitive units, between 3 to 80 repetitiveunits, or between 4 to 60 repetitive units, more preferably between 8 to48 repetitive units, or between 10 to 40 repetitive units and mostpreferably between 16 to 32 repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80repetitive units, each comprising at least one, preferably one,consensus sequence selected from the group consisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from A, S, G, Y, P, and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from Y, P, R, S, A, T, N and Q, more        preferably in each case independently selected from Y, P and Q;        and    -   iii) A_(x), wherein x is an integer from 5 to 10.

It is also preferred that the silk polypeptide comprises, essentiallyconsists of, or consists of between 2 to 80 repetitive units, between 3to 80 repetitive units, or between 4 to 60 repetitive units, morepreferably between 8 to 48 repetitive units, or between 10 to 40repetitive units and most preferably between 16 to 32 repetitive units,each comprising at least one, preferably one, amino acid sequenceselected from the group consisting of: GGRPSDTYG (SEQ ID NO: 18) andGGRPSSSYG (SEQ ID NO: 19).

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides according to the present invention areidentical repetitive units.

As to the silk polypeptide definitions, repetitive unit definitions,specific silk polypeptides, specific motifs and motif combinations, itis referred to the first aspect of the present invention.

It is preferred that the repetitive units are independently selectedfrom module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQID NO: 22), module K (SEQ ID NO: 23), module sp (SEQ ID NO: 24), moduleS (SEQ ID NO: 25), module R (SEQ ID NO: 26), module X (SEQ ID NO: 27),or module Y (SEQ ID NO: 28), or variants thereof (i.e. module Avariants, module C variants, module Q variants, module K variants,module sp variants, module S variants, module R variants, module Xvariants or module Y variants).

It is further preferred that the repetitive units are independentlyselected from module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).

It is particularly preferred that the repetitive units of the silkpolypeptide, preferably spider silk polypeptide, are independentlyselected from module A (SEQ ID NO: 20) or variants thereof, module C(SEQ ID NO: 21) or variants thereof, module Q (SEQ ID NO: 22) orvariants thereof, module K (SEQ ID NO: 23) or variants thereof, modulesp (SEQ ID NO: 24) or variants thereof, module S (SEQ ID NO: 25) orvariants thereof, module R (SEQ ID NO: 26) or variants thereof, module X(SEQ ID NO: 27) or variants thereof, module Y (SEQ ID NO: 28) orvariants thereof, module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides according to the present invention areidentical repetitive units.

In more preferred embodiments, the silk polypeptide according to thepresent invention comprises, essentially consists of, or consists ofbetween 2 to 80, between 3 to 80 repetitive units, or between 4 to 60repetitive units, preferably between 8 to 48 repetitive units, orbetween 10 to 40 repetitive units and most preferably between 16 to 32repetitive units, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units, which areindependently selected from module A (SEQ ID NO: or variants thereof,module C (SEQ ID NO: 21) or variants thereof, module Q (SEQ ID NO: 22)or variants thereof, module K (SEQ ID NO: 23) or variants thereof,module sp (SEQ ID NO: 24) or variants thereof, module S (SEQ ID NO: 25)or variants thereof, module R (SEQ ID NO: 26) or variants thereof,module X (SEQ ID NO: 27) or variants thereof, module Y (SEQ ID NO: 28)or variants thereof, module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ IDNO: 30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32),module C^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34).

Again, it should be noted that at least two of the repetitive unitscomprised in the silk polypeptides according to the present inventionare identical repetitive units.

As to the specific module combinations and module variant or fragmentdefinitions, it is referred to the first aspect of the presentinvention.

In further specific embodiments, the silk polypeptide, preferablyspidersilk polypeptide, further comprises one or more non-repetitive(NR) units.

More preferably, the NR unit is independently selected from the groupconsisting of NR3 (SEQ ID NO: 41 and SEQ ID NO: 45) or variants thereofand NR4 (SEQ ID NO: 42 and SEQ ID NO: 46) or variants thereof.

In preferred embodiments of the invention, the silk polypeptide,preferably spider silk polypeptide, is selected from the groupconsisting of ADF-3 (SEQ ID NO: 1 and SEQ ID NO: 47) or variantsthereof, ADF-4 (SEQ ID NO: 2 and SEQ ID NO: 48) or variants thereof,MaSp I (SEQ ID NO: 43 and SEQ ID NOs: 53-64) or variants thereof, MaSpII (SEQ ID NO: 44 and SEQ ID NOs: 65-78) or variants thereof,(C)_(m)NR_(z), NR_(z)(C)_(m), (AQ)_(n)NR_(z), NR_(z)(AQ)_(n),NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z), (C)_(m), (AQ)_(n), (QAQ)_(o), Y_(p),X_(p), and K_(p), wherein m is an integer of 8 to 48 (i.e. 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,or 48), n is an integer of 6 to 24 (i.e. 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24), o is an integer of 8 to 16(i.e. 8, 9, 10, 11, 12, 13, 14, 15, or 16), p is an integer of 8 to 16(i.e. 8, 9, 10, 11, 12, 13, 14, 15, or 16), z is an integer of 1 to 3(i.e. 1, 2, or 3), and NR stands for a non-repetitive unit.

More preferably, the silk polypeptide, preferably spider silkpolypeptide, is C₁₆, C₃₂, (AQ)₁₂, (AQ)₂₄, C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3,(AQ)₂₄NR3, Y₈, Y₁₆, X₈, X₁₆, K₈, or K₁₆.

As to the specific module combinations, NR3, NR4, ADF-3, ADF-4, MaSp Iand MaSp II variant or fragment definitions, it is referred to the firstaspect of the present invention.

In further preferred embodiments of the invention, the compound is apharmaceutically active compound, a cosmetic substance, an agriculturalsubstance, a chemoattractant, a chemorepellent, an anti-fungalsubstance, an anti-bacterial substance, a nutrient, a dietarysupplement, a dye, a fragrance or an agent selected from the groupconsisting of hemostatic agents, growth stimulating agents, inflammatoryagents, anti-fouling agents, antimicrobial agents and UV protectingagents.

In further specific embodiments, the compound has an overall positivenet charge. In further specific embodiments, the compound is able topermeate into the silk matrix, preferably spider silk matrix, byelectrostatic interaction and/or diffusion. Preferably, the compound hasan overall positive net charge and is able to permeate into the silkmatrix, preferably spider silk matrix, by electrostatic interactionand/or diffusion.

In further preferred embodiments, the compound has a neutral or alkalinenature. Preferably, the compound has an overall positive net charge, isable to permeate into the silk matrix, preferably spider silk matrix, byelectrostatic interaction and/or diffusion and has a neutral or alkalinenature.

In preferred embodiments of the invention, the compound is released fromthe silk particles, preferably spider silk particles, by diffusion uponexposure to physiological conditions. The silk particles, preferablyspider silk particles, according to present invention are, therefore,clearly distinguishable from the silk particles, e.g. spider silkparticles, of the prior art, where release of the encapsulated compoundis dependent on proteolysis. The compound is capable of being releasedupon exposure of the loaded silk particles, preferably spider silkparticles, to physiological conditions, i.e. introducing the silkparticles, preferably spider silk particles, into a buffer or an aqueoussolution. Preferably the silk particles, more preferably spider silkparticles, show a sustained and controlled release of the loadedcompound. Sustained (or controlled) release refers to the gradualrelease of a compound from the silk matrix, preferably spider silkmatrix, over a period of time. While there may be an initial burstphase, it is preferred that the release display relatively linearkinetics, thereby providing a constant supply of the compound over therelease period. The release period may vary from several hours toseveral months, depending upon the properties of the compound and itsintended use. For example, it can be desirable that the cumulativerelease of a pharmaceutically active compound from the silk matrix,preferably spider silk matrix, over a certain treatment period berelatively high to avoid the need for excessive loading of the matrixand consequent waste of unreleased pharmaceutically active agent.

Preferably, the release profile of the silk particles, preferably spidersilk particles, has a small burst release within the first 24 hours. Infurther preferred embodiments, less than 20%, preferably less than 15%,and most preferably less than 10%, e.g. less than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, or 10%, of the compound is released, e.g.into the surrounding medium, within the first 24 hours. Said surroundingmedium may be a buffered solution, a physiological buffered solution,blood, a body fluid, lymph, liquor, or water.

Preferably, up to 100% of the compound is released, e.g. into thesurrounding medium, within 36 hours, 48 hours, or 72 hours, morepreferably within 7 days, 14 days, 21 days, 31 days, or 35 days, mostpreferably within 5 weeks, 6 weeks, 7 weeks, or 8 weeks. As shown inexample 8, almost 100% of the compound ethacridine lactate is releasedwithin 35 days.

In a third aspect, the invention relates to a pharmaceutical compositioncomprising the silk particles, preferably spider silk particles,according to the invention and additionally a pharmaceuticallyacceptable buffer, diluent and/or excipient, wherein the pharmaceuticalcomposition is being useful for controlled and sustained delivery, andwherein the compound is a pharmaceutically active compound.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising the silk particles, preferably spider silkparticles, according to the invention and additionally one or morepharmaceutically acceptable buffer(s), diluent(s) and/or excipient(s).Preferably, the pharmaceutical composition is (useful) for controlledand sustained delivery of a compound. It is further preferred that thesilk particle, preferably spider silk particle, of the inventioncomprises a compound which is a pharmaceutically active compound.

The compound mentioned above can be any pharmaceutically compound asmentioned above. In a preferred embodiment of the invention, thecompound has a molecular weight of 50 Da or about 50 Da to 20 kDa orabout 20 kDa; or 50 Da or about 50 Da to 10 kDa or about 10 kDa,preferably 50 Da or about 50 Da to 6 kDa or about 6 kDa, more preferably50 Da or about 50 Da to 4 kDa or about 4 kDa and most preferably 50 Daor about 50 Da to 1 kDa or about 1 kDa, e.g. 50 Da, 100 Da, 150 Da, 200Da, 250 Da, 300 Da, 350 Da, 400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650Da, 700 Da, 750 Da, 800 Da, 850 Da, 900 Da, 950 Da, 1 kDa, 1.5 kDa, 2kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5kDa, 7 kDa, 7.5 kDa, 8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa, 10 kDa, 11 kDa, 12kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, or 20 kDa.

As used herein, the terms “subject” or “patient” may be usedinterchangeably to refer to a mammal that may benefit from theadministration of a composition or method as recited herein. Most oftenthe subject or patient will be a human or other mammal such as forexample horses, dogs or cats.

“Administration” refers to the manner in which an active agent orcomposition containing such is presented to a subject. Thepharmaceutical composition according to the invention may beadministered to a subject using several ways.

Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,rectal, by inhalation, or topical, particularly to the ears, nose, eyes,or skin. Due the constant release profile of the silk particles,preferably spider silk particles, which are capable of releasing theloaded pharmaceutically over a period of weeks, the presentpharmaceutical composition is in particular well-suited for parenteraladministration. Since the silk particles, preferably spider silkparticles, are also gastro-resistant the pharmaceutical composition arehowever also eminently suitable for oral forms of administration. It isalso possible to formulate the silk particles, preferably spider silkparticles, loaded with a pharmaceutically active compound in a depotsystem. For example, the particles may be embedded in films, lipids orgels.

The present pharmaceutical composition can optionally comprise asuitable amount of a pharmaceutically acceptable excipient so as toprovide the form for proper administration to a subject. Suchpharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea and the like. In addition,auxiliary, stabilizing, thickening, lubricating, and coloring agents canbe used. Saline solutions and aqueous dextrose and glycerol solutionscan also be employed as liquid excipients, particularly for injectablesolutions. Suitable pharmaceutical excipients also include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or can contain pH bufferingagents. Further examples of suitable pharmaceutically acceptableexcipients described herein may be found in the “Handbook ofPharmaceutical Excipients”, 2nd Edition, (1994), Edited by A Wade and PJWeller. Acceptable carrier or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaroedit. 1985). Examples of suitable carriers include lactose, starch,glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol andthe like. Examples of suitable diluents include ethanol, glycerol andwater.

In a fourth aspect, the invention relates to a cosmetic compositioncomprising the silk particles, preferably spider silk particles,according to the invention for controlled and sustained delivery,wherein the compound is a cosmetic compound.

In another further aspect, the invention relates to a cosmeticcomposition comprising the silk particles, preferably spider silkparticles, according to the invention and additionally one or morecosmetically acceptable buffer(s), diluent(s) and/or excipient(s).Preferably, the cosmetic composition is (useful) for controlled andsustained delivery of a compound. It is further preferred that the silkparticle, preferably spider silk particle, of the invention comprises acompound which is a cosmetic compound.

In a fifth aspect, the invention relates to silk particles, preferablyspider silk particles, loaded with a compound, wherein the compound iswater soluble, has a molecular weight of 50 Da to 20 kDa and/or has anoverall positive net charge and wherein the silk particles, preferablyspider silk particles, comprise one or more silk polypeptides,preferably spider silk polypeptides, comprising at least two identicalrepetitive units, the particles being obtainable by a process accordingto the invention.

Further embodiments will become obvious from the following exampleswhich illustrate the invention in some of its major aspects, withoutlimiting the scope thereof.

Free Text of the Sequence Listing

SEQ ID NOs: 3, 20-24, 27-34, 45-96: SEQ ID NO: 3 GPGXX; X =A, S, G, Y, P, Q SEQ ID NO: 20 Modul A: GPYGPGASAA AAAAGGYGPG SGQQSEQ ID NO: 21 Modul C: GSSAAAAAAA ASGPGGYGPE NQGPSGPGGY GPGGPSEQ ID NO: 22 Modul Q: GPGQQGPGQQ GPGQQGPGQQ SEQ ID NO: 23Modul K: GPGGAGGPYGPGGAGGPYGPGGAGGPY SEQ ID NO: 24Modul sp: GGTTIIEDLD ITIDGADGPITISEELTI SEQ ID NO: 27Modul X: GGAGGAGGAG GSGGAGGS SEQ ID NO: 28Modul Y: GPGGAGPGGY GPGGSGPGGY GPGGSGPGGY SEQ ID NO: 29Modul A^(C): GPYGPGASAA AAAAGGYGPG CGQQ SEQ ID NO: 30Modul A^(K): GPYGPGASAA AAAAGGYGPG KGQQ SEQ ID NO: 31Modul C^(C): GSSAAAAAAA ASGPGGYGPE NQGPCGPGGY GPGGP SEQ ID NO: 32Modul C^(K1): GSSAAAAAAA ASGPGGYGPE NQGPKGPGG Y GPGGP SEQ ID NO: 33Modul C^(K2): GSSAAAAAAA ASGPGGYGPK NQGPSGPGGY GPGGP SEQ ID NO: 34Modul C^(KC): GSSAAAAAAA ASGPGGYGPK NQGPCGPGGY GPGGPSEQ ID NO: 45 - NR3 (ADF-3):MASMTGGQQMGRGSMGAASAAVSVGGYGPQSSSAPVASAAASRLSSPAASSRVSSAVSSLVSSGPTNQAALSNTISSVVSQVSASNPGLSGCDVLVQALLEVVSALVSILGSSSIGQINYGASAQYTQMVGQSVAQALAG SEQ ID NO: 46 - NR4 (ADF-4):MASMTGGQQMGRGSMGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVALLSSASIGQVNVSSVSQSTQM ISQALSGSEQ ID NO: 47 - ADF-3:MASMTGGQQMGRDPNSARAGSGQQGPGQQGPGQQGPGQQGPYGPGASAAAAAAGGYGPGSGQQGPSQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGSGQQGPGGQGPYGGSSAAAAAAGGNGPGSGQQGPGQQGPGQQGPGASAAAAAAGGYGPGSGQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGYGQQGPGQQGPGGQGPYGPGASAASAASGGYGPGSGQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGGQGPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGGQGAYGPGASAAAGAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPGQQGPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPVGQGPYGPGAASAAVSVGGYGPQSSSAPVASAAASRLSSPAASSRVSSAVSSLVSSGPTNQAALSNTISSVVSQVSASNPGLSGCDVLVQALLEVVSALVSILGSSSIGQINYGASAQYTQMVGQSVAQALA SEQ ID NO: 48 - ADF-4:MASMTGGQQMGRAARAGSSAAAAAAASGSGGYGPENQGPSGPVAYGPGGPVSSAAAAAAAGSGPGGYGPENQGPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGPSGPGASSAAAAAAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGPSGPGGPGASAAAAAAAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAAAAAAGSGPGGYGPGNQGPSGPGGYGPGGPGSSAAAAAAASGPGGYGPGSQGPSGPGVYGPGGPGSSAAAAAAAGSGPGGYGPGNQGPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGPSGPGASSAAAAAAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAAAAAASGPGGYGPGSQGPSGPGGSRGYGPGSQGPGGPGASAAAAAAAAASGPGGYGPGSQGPSGPGYQGPSGPGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALSG Araneus diadematus fibroinSEQ ID No 49: >gi|1263283|gb|AAC47008.1| fibroin-1HESSYAAAMAASTRNSDFIRNMSYQMGRLLSNAGAITESTASSAASSASSTVTESIRTYGPAAIFSGAGAGAGVGVGGAGGYGQGYGAGAGAGAGAGAGAGGAGGYGQGYGAGAAAAAGAGAGAAGGYGGGSGAGAGGAGGYGQGYGAGSGAGAGAAAAAGASAGAAGGYGGGAGVGAGAGAGAAGGYGQSYGSGAGAGAGAGAAAAAGAGARAAGGYGGGYGAGAGAGAGAAASAGASGGYGGGYGGGAGAGAVAGASAGSYGGAVNRLSSAGAASRVSSNVAAIASAGAAALPNVISNIYSGVLSSGVSSSEALIQALLEVISALIHVLGSASIGNVSSVGVNSALNAVQNAVGAYAG SEQ ID No 50: >gi|1263285|gb|AAC47009.1| fibroin-2GSQGAGGAGQGGYGAGGGGAAAAAAAAVGAGGGGQGGLGSGGAGQGYGAGLGGQGGASAAAAAAGGQGGQGGQGGYGGLGSQGAGGAGQLGYGAGQESAAAAAAAAGGAGGGGQGGLGAGGAGQGYGAAGLGGQGGAGQGGGSGAAAAAGGQGGQGGYGGLGPQGAGGAGQGGYGGGSLQYGGQGQAQAAAASAAASRLSSPSAAARVSSAVSLVSNGGPTSPAALSSSISNVVSQISASNPGLSGCDILVQALLEIISALVHILGSANIGPVNSSSAGQSASIVGQSVYRALS SEQ ID No 51: >gi|1263287|gb|AAC47010.1| fibroin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 ID No 52: >gi|1263289|gb|AAC47011.1| fibroin-4AGSSAAAAAAASGSGGYGPENQGPSGPVAYGPGGPVSSAAAAAAAGSGPGGYGPENQGPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGASGPGGPGASAAAAAAAAAASGPGGYGPGSQGPSGPGAYGPGGPGSSAAAAAAAASGPGGYGPGSQGPSGPGVYGPGGPGSSAAAAAAAGSGPGGYGPENQGPSGPGGYGPGGSGSSAAAAAAAASGPGGYGPGSQGPSGPGGSGGYGPGSQGGSGPGASAAAAAAAASGPGGYGPGSQGPSGPGYQGPSGPGAYGPSPSASASVAASVYLRLQPRLEVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALSmajor ampullate spidroin 1 SEQ ID No 53: >gi|185179256|gb|ACC77633.1|major ampullate spidroin 1 [Nephila clavipes]AGQGGLGGQGAGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGAGQGGYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAAAAGGAGQGGYGGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEVVSALIHILGSSSIGQVNYGSAGQATQIVGQSVYQALGSEQ ID No 54: >gi|50363145|gb|AAT75312.1|major ampullate spidroin 1 [Nephila clavipes]GGQGAGRGAGAAAAAAGGAGQGGYGGLGGQGAGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGYGGQGAEAAAAAAAGGAAQGGQGLGGQGAAAAAGGAGQGGEGGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGAGAAAAAAGGAGQGGYGGLGGQGAGRGAGAAAAAAGGAAQGGYGDLGSQGAGAAAAAAGSAGQGGYGGLGGQGAGQGAGAAAAAAGSAGQGGLGGRAGQGAGAASAAAGGAGQGGYGGLGGQGAGQGGYGGVGSGASAASSAASRLSSPEASSRVSSAVSNLVSSGPTNSAALSSTISNVVSQIGASNPGLSGCDVLVQALLEVVSALIHILGSSSIGQVNYGSAGQATQIVGQSIYQALG SEQ ID No 55: >gi|50363143|gb|AAT75311.1|major ampullate spidroin 1 [Nephila clavipes]AGAAAAAGSAGQGGYGGQGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGGAGQGGLGGQGAGQGAGAAAAAAGGAGQGGYGGLGNQGAGRGGQGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGSQGSGRGGLGGQGAGAAAAAAGGAGQGGLGGQGAGQGAGAAAAAAGGVRQGGYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEVVSALIHILGSSSIGQVNYGSAGQATQIVGQSVYQALGSEQ ID No 56: >gi|50363141|gb|AAT75310.1|major ampullate spidroin 1 [Nephila clavipes]GGLGIQGSGRGGLGGQGAVAAAAAAAGGAVQVVLGGQGAGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGQGAGARTAAAVGAGQGGYGGQGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGSAEQGLGGQGAGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGSQGSGRGGLGGQGAGAAAAAAGGAGQGGLGGQGAGQGAGAAAAAAGGVRQGGYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEVVSALIHILGSSSIGQVNYGSAGQATQIVGQSVYQALG SEQ ID No. 57: >gi|50363139|gb|AAT75309.1|major ampullate spidroin 1 [Nephila clavipes]QGTDAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGSQGSGRGGLGGQGAGAAAAAAGGAGQGAGQGAGAAAAAAGGVRQGGYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGSSNPGLSGCDVLIQALLEVVSALIQILGSSSIGQVNYGSAGQATQIVGQSVYQALGSEQ ID No. 58: >gi|50363137|gb|AAT75308.1|major ampullate spidroin 1 [Nephila clavipes]GQGAGQGAGAAAAAAGGAGQGGYGGLGNQGAGRGGQGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGAGQGGYGGLGIQGSGRGGLGGQGAGAAAAAAGGAGQGGLGGQGAGQGAGAAAAAAGGVRQGGYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGoQGAGAAAAGGAGQGGYGGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEVVSALIQILGSSSIGQVNYGSAGQATQIVGQSVYQALGSEQ ID No. 59: >gi|13562006|gb|AAK30606.1|AF350277_1 major ampullate spidroin 1[Nephila madagascariensis]GLGGQGAGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGYGGQGAGAAAAAAAGGAGQGGYGGLGSWAGQGGYGGLGGQGAGQGAAAAAAAGGAGQGGYGGLGSQGAGRGGYGGQGAGAAAAATGGAGQGGYGGVGSGASAASAAASRLSSpQASSRVSSAVSNLVASGPTNSAALSSTISNAVSQIGASNPGLSGCDVLIQALLEVVSALIHILGSSSIGQVNYGSAGQATQSEQ ID No. 60: >gi|13562022|gb|AAK30614.1|AF350285_1 major ampullate spidroin 1[Tetragnatha kauaiensis]SGLGGAGQGAGQGASAAAAAAAXGGLGGGQGAGQGGQQGAGQGGYGSGLGGAGQGASAAAAAAAAGGLGGGQGAGQGGQQGAGQGGYGSGLGGAGQGASAAAAAAAAGGLGGGQGAGQGGINGAGQGGYGSGLGGAGQGAGQGASAAAAAAAGGLGGGQGGYGSGLGGVGQGGQGALGGSRNSATNAISNSASNAVSLLSSPASNARISSAVSALASGAASGPGYLSSVISNVVSQVSSNSGGLVGCDTLVQALLEAAAALVHVLASSSGGQVNLNTAGYTSQLSEQ ID No. 61: >gi|13562010|gb|AAK30608.1|AF350279_1 major ampullate spidroin 1[Nephila senegalensis]GLGGQGAGRGAGAAAAAAGGAGQGGYGGLGGQGAGAAAAAAGGAGQGGQGLGGRGAAAAGGAGQGGYGGLGGQGAGRGAGAAAAAAGGAGQGGYGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGYGGQGAGAAVAAIGGVGQGGYGGVGSGASAASAAASRLSSPEASSRVSSAVSNLVSSGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEVVSALVHILGSSSIGQVNYGSAGQATQSEQ ID No. 62: >gi|13582024|gb|AAK30615.1|AF350286_1 major ampullate spidroin 1[Tetragnaiha versicolor]SGQGASAAAAAAGGLGGGQGGYGSGIZGAGQGGQQGAGQGAAAAAASAAAGGLGGGQGGQQGAGRGGLQGAGQGGQGALGGSRNSAANAVSRLSSPASNARISSAVSALASGGASSPGYLSSIISNVVSQVSSNNDGLSGCDTVVQALLEVAAALVHVLASSNIGQVNLNTAGYTSQLSEQ ID No. 63: >gi|13561998|gb|AAK30602.1|AF350273_1 major ampullate spidroin 1[Latrodectus geometricus]AGSGQGGYGQGYGEGGAGQGGAGAAAAAAAAAGGAGQGGQGGYGQGYGQGGAGQGGAGAAAAAAAGGAGQGGYGRGGAGQGAAAAAAAAGSGQGGQGGYGQGYGQGGAGQGGAGAAAAAAAAGGAGQGGYGRGGAGQGGAAAAAAAAGGAGQGGQGGYGQGYGQGGAGQGGAGAAAAAAAAGGAGQGGYGRGGAGQGGSAAAAAAAGGAGQGGYGRGGAGQGGAGSAAAAAAAGGSGQGGQGGYGQGYGQGGAGQGGAAAAASALAAPATSARISSHASTLLSNGPTNPASISNVISNAVSQISSSNPGASSCDVLVQALLELVTALLTIIGSSNVGNVNYDSSGQYAQVVSQSVQNAFVSEQ ID No. 64: >gi|13561984|gb|AAK30595.1|AF350266_1 major ampullate spidroin 1[Argiope trifasciata]AAAAAAAAAGGQGGQGGYDGLGSQGAGQGGYGQGGAAAAAAAASGAGSAQRGGLGAGGAGQGYGAGSGGQGGAGQGGAAAATAAAAGGQGGQGGYGGLGSQGSGQGGYGQGGAAAAAAAASGDGGAGQEGLGAGGAGQGYGAGLGGQGGAGQGGAAAAAAAAAGGQGGQGGYGGLGSQGAGQGGYGQGGAAAAAAAASGAGGAGQGGLGAAGAGQGYGAGSGGQGGAGQGGAAAAAAAAAGGQGGQGGYGGLGSQGAGQGGYGQGGVAAAAAAASGAGGAGRGGLGAGGAGQEYGAVSGGQGGAGQGGEAAAAAAAAGGQGGQGGYGGLGSQGAGQGGYGQGGAAAAAAAASGAGGARRGGLGAGGAGQGYGAGLGGQGGAGQGSASAAAAAAAGGQGGQGGyGGLGSQGSGQGGYGQGGAAAAAAAASGAGGAGRGSLGAGGAGQGYGAGLGGQGGAGQGGAAAAASAAAGGQGGQGGYGGLGSQGAGQGGYGQGGAAAAAASAGGQGGQGGYGGLGSQGAGQGGYGGGAFSGQQGGAASVATASAAASRLSSPGAASRVSSAVTSLVSSGGPTNSAALSNTISNVVSQISSSNPGLSGCDVLVQALLEIVSALVHILGSANIGQVNSSGVGRSASIVGQSINQAFS major ampullate spidroin 2SEQ ID No. 65: >gi|50363155|gb|AAT75317.1|major ampullate spidroin 2 [Nephila clavipes]GGYGPGQQGPGGYGPGQQGPSGSGSAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAAGPGGYGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAF SEQ ID No. 66: >gi|50363153|gb|AAT75316.1|major ampullate spidroin 2 [Nephila clavipes]PGGYGPGQQGPSGAGSAAAAAAAGPGQQGLGGYGPGQQGPGGYGPGQQGPGGYGPGSASAAAAAAGPGQQGPGGYGPGQQGPSGPGSASAAAAAAAAGPGGYGPGQQGPGGYAPGQQGPSGPGSAAAAAAAAAGPGGYGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAF SEQ ID No. 67: >gi|50363147|gb|AAT75313.1|major ampullate spidroin 2 [Nephila clavipes]GPGGYRPGQQGPSGPGSAAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPSGAGSAAAAAAAGPGQQGLGGYGPGQQGPGGYGPGQQGPGGYGPGSASAAAAAAGPGQQGPGGYGPGQQGPSGPGSASAAAAAAGPGGYGPGQQGPGGYAPGQQGPSGPGSAAAAAAAARAGPGGYGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAFSEQ ID No. 68: >gi|50363151|gb|AAT75315.1|major ampullate spidroin 2 [Nephila clavipes]GRGAGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGPGQQGPGGYGPGQQGPGGYGPGQQSPSGPGSAAAAAAAAAGPGQQGPGGYGPGQQGPSGPGSASAAAAAAGPGGYGPGQQGPGGYAPGQQGPSGPGSAAAAAAARAGPGGYGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAFSEQ ID NQ. 69: >gi|50363149|gb|AAT75314.1|major ampullate spidroin 2 [Nephila clavipes]SAAAAAAAAAGPGGYGPGQQGPGGYGPGQQGPSGAGSAAAAAGPGQQGLGGYGPGQQGPGGYGPGQQGPGGYGPGSASAAAAAAGPGQQGPGGYGPGQQGPSGPGSASAAAAAAGPGGYGPGQQGPGGYAPGQQGPSGPGSAAAAAAAAAGPGGYGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAFSEQ ID No. 70: >gi|13562012|gb|AAK30609.1|AF350280_1 major ampullate spidroin 2[Nephila senegalensis]QGPGGYGPSGPGSAAAASAAAGPGQQGPGAYGPSGPGSAAAAAGPGXYGPGQQGPSGPGAAAAAAGPGQ0GPGGYGPGAAAAAAAAAGPGQQGPVAYGPSGPGSAASAAGPGGYGPARYGPSGSAAAAAAAGAGSAGYGPGPQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVIXNAVSQIGASNPGLSGCDVLIXALLEIVSACVTILSSSSIGQVNYGAASEQ ID No. 71: >gi|13562008|gb|AAX30607.1|AF350278_1 major ampullate spidroin 2[Nephila madagascariensis]QGPSGPGSAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAAAGPGQQGPGGYGPGPQGPGGYGPGQQGPSGYGPGQQGPSGPGSAASAAAAAGSGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAAGPGQQGPGGYGPGQQGPGQQGPSGPGSAAAAAAAGPGPQGPGGYGPGQQGPGGYGPSGPGSAAAAAAAAGPGQQGPGGYGPGQQRPSGYGPGQQGPSGPGSAAAAAAAGPGQQGPGAYGPSGPGSAAAAAGLGGYGPAQQGPSGAGSAAAAAAAGPGGYGPVQQGPSGPGSAAGPGGYGPAQQGPARYGPGSAAAAAAAAGSAGYGPGPQASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSACVTILSSSSIGQVNYGAASEQ ID No. 72: >gi|13562062|gb|AAK30604.1|AF350275_1 major ampullate spidroin 2[Latrodectus geometricus]AGPGSYGPSGPGGSGAAAAAAAASGPGGQQGYGPGGPGASAAAAAAAGGSGPGGYGQGPSGYGPSGPGAQQGYGPGGQGGSGAAAAAAAAAGSGPGGYGPGAAGPGNYGPSGPGGSGAAASAAAASGPGGQQGYGPGGSGAAAAAASGGAGPGRQQGYGPGGSGAAAAAAAAXGGSGPGGYGQGPXGYGPGGQGGSGGAAAAAAAASSGPXGYGPGAAGPGNYGPSGPGGSGAAAAAAAASGPGGQQGYGPGGSGASAAAAAGGAGXGRQQAYGPGGSGAAAAAASGSGGYGPAQYGXSSVASSAASAASALSSPTTHARISSHASTLLSSGPTNSAAISNVISNAVSQVSASNPGSSSCDVLVQALLELITALISIVDSSNIGQVNYGSSGQYAQMVGSEQ ID No. 73: >gi|13561986|gb|AAK30596.1|AF350267_1 major ampullate spidroin 2[Argiope trifasciata]AGPGYGPGAGQQGPGSQGPGSGGQQGPGGQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSGGQQGGQGSGQQGPGGAGQGGPRGQGPYGPGAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQGPGSGGQQGPGGQGPYGPSDAAAAAAAGPGYGPGAGQQGPGSGGQQGGQGSGQQGPGGAGQGGPRGQGPYGPGAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQAPVASAAASRLSSPQASSRVSSAVSTLVSSGPTNPASLSNAISSVVSQVSSSNPGLSGCDVLVQALLEIVSALVHILGSSSIGQINYAASSQYAQLVGQSLTQALGSEQ ID No. 74: >gi|13561978|gb|AAX30592.1|AF350263_1 major ampullate spidroin 2[Argiope aurantia]PGGAGQQGPGGQGPYGPGAAAAAAAAGGYGPGAGQQGPXGAGQQGPGSQGPGGAGQQGPGGQGPYGPGAAAAAAAVGGYGPGAGQQGPGSQGPGSGGQQGPGGQGPYGPSAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGGLGPYGPSAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQRPGGLGPYGPSAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQRPGGLGPYGPSAAAAAAAAGGYGPGAGQQGPGSQAPVASAAASRLSSPQASSRVSSAVSTLVSSGPTNPAALSNAISSVVSQVSASNPGLSGCDVLVQALLELVSALVHILGSSSIGQINYAASSEQ ID No. 75: >gi|70913274|gb|AAZ15372.1|major ampullate spidroin 2 [Argiope trifasciata]GQGSGQQRPGGAGQGGLGPYGPGAAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGSRGPYGPSAAAAAAAAGPGYGPGAGQRGPRSQGPGSGGQQGPGGQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQAPVASAAASRLSSPQASSRVSSAVSTLVSSGPTNPASLSNAISSVVSQVSASNPGLSGCDVLVQALLEIVSALVHILGSSSIGQINYAASSQYAQMVG SEQ ID No. 76: >gi|70913273|gb|AAZ15371.1|major ampullate spidroin 2 [Argiope trifasciata]MNWSIRLALLGFVVLSTQTVFSAGQGATPWENSQLAESFISRFLRFIGQSGAFSPNQLDDMSSIGDTLKTAIEKMAQSRKSSKSKLQALNMAFASSMAEIAVAEQGGLSLEAKTNAIASALSAAFLETTGYVNQQFVNEIKTLIFMIAQASSNEISGSAAAAGGSSGGGGGSGQGGYGQGAYASASAAAAYGSAPQGTGGPASQGPSQQGPVSQPSYGPSATVAVTAVGGRPQGPSAPRQQGPSQQGPGQQGPGGRGPYGPSAAAAAAAAGGYGPGAGQQGQQAGQGSGQQGPGGAGQGGPRGQGPYGPGAATAAAAAAGPGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQGPRSGGQQGPGGQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSGGQQGGPGSGQQGPGGAGQGGPRGQGPYGPGAAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSQGPGSGGQQGPGGQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSGGQQGGQGSGQQGPGGAGQGGPRGQGPYGPGAAAAAAAAAGGYGPGAGQQGPGSQGPGSGGQQGPGSQGPYGPSAAAAAAAAGPGYGPGAGQQGPGSGGQQSEQ ID No. 77: >gi|164709224|gb|ABY67417.1| major ampullate spidroin 2[Latrodectus geometricus]LRWSSKDNADRFINAFLQAASNSGAFSSDQVDDMSVIGNTLMTAMDNMGGRITPSKLQALDMAFASSVAEIAVALGQNVGGATNAISNALRSAFYQTTGVVNNQFISEISNLINMFAQVSANEVSYASGGSSSAAASAAASAGPAAQQVYAPSAGAPAAATASSGPGAYGPSAPGGPSAAAAAAASGGAGPGRQQSYGPGGSGAAAAAAATGGSGPGGYGQGPASYAPSGPGGQQGYGPGGSGAASAAAAAASSGPGGYGPGASGPGSYGPSGPGGSGAAAAAAAASAPGGQQGYGPGGSGAAAAAAAGGAGPGSQQAYGPGGSGAAAAAAAGPGSGGQQGYGPGGSAAAAAAAAAGGSGPGGYGQGPAGYGPSGPGAQQGYGPGGPGSEQ ID No. 78: >gi|13561996|gb|AAK30601.1|AF350272_1 major ampullate spidroin 2[Gasteracantha mammosa]GQQGPGSQGPYGPGAAAAAAAAAGGYRPVSGQQGPGQQGPGSGGQQGPGGQRPYGPGAAAAAAAAGGYGPGSGQGGPGQQGPGSGGQQGPGGQGPYGPGAAAAAAAAAGGYGPGSGQGGQQGPGSQGPGSGGQ0GPGGQGPYGPSAAAAAAAVGGYGPGAGQQGPGQQGPGSGGQRGPGGQGPYGPGAAAAAAAAAGGYGPASGQQGPGQQGPGSGGQRGPGGQGPYGPGAAAAASAGGYGPGSGGSPASGAASRLSSPQAGARVSSAVSALVASGPTSPAAVSSAISNVASQISASNPGLSGCDVLVQALLEIVSALVSILSSASIGQINYGASGQYAAMIminor ampullate silk protein SEQ ID No. 79: >gi|2605800|gb|AAC14590.1|minor ampullate silk protein [Nephila clavipes]GAGGYGRGAGAGAAAVAGADAGGYGRNYGAGTTAYAGARAGGAGGYGGQGGYSSGAGAAAASGAGADITSGYGRGVGAGAGAETIGAGGYGGGAGSGARAASASGAGTGYGSSGGYNVGTGISTSSGAASSYSVSAGGYASTGVGIGSTVTSTTSRLSSAEACSRISAAASTLVSGSLNTAALPSVISDLFAQVSASSPGVSGNEVLIQVLLEIVSSLIHILSSSSVGQVDFSSVGSSAAAVGQSMQVVMGSEQ ID No. 80: >gi|2605802|gb|AAC14591.1|minor ampullate silk protein MiSp2 [Nephila clavipes]SYGPSVMPTSAGSYGAGAGGFGAGASAGVGAGAGTVAGYGGQGGYGAGSAGGYGRGTGAGAAAGAGAGATAGAGAGAAAGAGAGAGNSGGYSAGVGVGAAAAAAGGGAGTVGGYGRGAGVGAGAAAGFAAGAGGAGGYRRDGGYGAGAGAGAAAA SEQ ID No. 81: >gi|2605798|gb|AAC14589.1|minor ampullate silk protein MiSp1 [Nephila clavipes]RGAASGAGAAAGAGAGAGGAGYGGQIGYGAGAGAGAAAAAGAGAGGAAGYGRGAGAGSGAAAGAGSGAGAGGYGGQAGYGAGAGAGSSAGNAFAQSLSSNLLSSGDFVQMISSTTSTDHAVSVATSVAQNVGSQLGLDANAMNNLLGAVSGYVSTLGNAISDASAYANALSSAIGNVLANSGSISESTASSAASSAASSVTTTLTSYGPAVFYAPSASSGGYGAGAGAVAAAGAAGAGGYGRGAGGYGGQGGYGAGAGAGAAAAAGAGAGGAGGYGRGAGAGAGAAAGAGAGAGGAGYGGQGGYGAGAGAGAAAAAGAGAGGAGGYGRGAGAGAGAAAGAGAGGYGGQGGYGAGAGAGAAAAAAGAGSGGAGGYGRGAGAGAGAAAGAGAGAGSYGGQGGYGAGAGAGAAAAAGAGAGAGGYGRGAGAGAGAGAGAAARAGAGAGGAGYGGQGGYGAGAGAGAAAAAGAGAGGAGGYGRGAGAGAGAAAGAGAGAGGYGGQSGYGAGAGAAAAAGAGAGGAGGYGRGAGAGAGAAAGAGAGAAAGAGAGGYGGQGGYGAGAGAGAAAAAGAGAGGAGGYGRGAGAGAGVAAGAGAGGYGGQGGYGAGAGAGAAAAAATGAGGAGGYGRGAGAGAGAAAGAGAGTGGAGYGGQGGYGAGAGAGAAAAAGAGAGGAGYGRGAGAGAGAAAGAGAGAAAGAGAGAGGYGGQGGYGAGAGAGAAAAAGAGAGGAAGYSRGGRAGAAGAGAGAAAGAGAGAGGYGGQGGYGAGAGAGAAAAAGAGSGGAGGYGRGAGAGAAAGAGAAAGAGAGAGGYGGQGGYGAGAGAAAAAGAGAGRGGYGRGAGAGGYGGQGGYGAGAGAGAAAAAGAGAGGYGDKEIACWSRCRYTVASTTSRLSSAEASSRISSAASTLVSGGYLNTAALPSVISDLFAQVGASSPGVSDSEVLIQVLLEIVSSLIHILSSSSVGQVDFSSVGSSAAAVGQSMQVVMG flagelliform silk proteinSEQ ID No. 82: >gi|2833649|gb|AAC38847.1|flagelliform silk-protein [Nephila clavipes]GPGGVGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGYGPGGSGPGGYGPGGTGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGpGGAGPGGAGGAGGAGGAGGSGGAGGSGGTTIIEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGSGPGGYGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGSGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGTGPGGTGPGGSGPGGYGPGGSGPGGSGPGGSGPGGYGPSGSGPGGYGPSGSGPGGYGPGGSGPGGYGPGGSGAGGTGPGGAGGAGGAGGSGGAGGSGGAGGSGGAGGSGGVGGSGGTTITEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGVSGPGGVGPGGSGPGGVGSGGSGPGGVGPGGYGPGGSGSGGVGPGGYGPGGSGGFYGPGGSEGPYGPSGTYGSGGGYGPGGAGGPYGPGSPGGAYGPGSPGGAYYPSSRVPDMVNGIMSAMQGSGFNYQMFGNMLSQYSSGSGTCNPNNVNVLMDALLAALHCLSNHGSSSFAPSPTPAAMSAYSNSVGRMFAYSEQ ID No. 83: >gi|2833647|gb|AAC38846.1|flagelliform silk protein [Nephila clavipes]MGKGRHDTKAKAKAMQVALASSIAELVIAESSGGDVQRKTNVISNALRNALMSTTGSPNEEFVHEVQDLIQMLSQEQINEVDTSGPGQYYRSSSSGGGGGGQGGPVVTETLTVTVGGSGGGQPSGAGPSGTGGYAPTGYAPSGSGAGGVRPSASGPSGSGPSGGSRPSSSGPSGTRPSPNGASGSSPGGIAPGGSNSGGAGVSGATGGPASSGSYGPGSTGGTYGPSGGSEPFGPGVAGGPYSPGGAGPGGAGGAYGPGGVGTGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPdGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGTGPGGYGPGGTGPGGVGPGGAGPGGYGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGYGPGGSGPGGAGPSGAGLGGAGPGGAGLGGAGPGGAGTSGAGPGGAGPGGAGQGDAGPGGAGRGGAGRGGVGRGGAGRGGAGRGGARGAGGAGGAGGAGGSGGTTIVEDLDITIDGADGPITISEELTIGGAGAGGSGPGGAGPGNVGPGRSGPGGVGPGGSGPGGVGPGSFGPGGVGPGGSGPGGVGSGGSGQGGVRPSGSGPGGVGTGGVGPGGAGGPYGPGGSGPGSAGSAGGTYGPGGFGGPGGFGGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGSYGLGGAGGSGGVGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGSGGYGPGGSGPGGSGPGGYGPGGTGPGGSESGGYGPGGSGPGGSGPGGSGPGGSGPGGYGPGGSGPSSFVPGGSGPGGSGPGGAGPGGAGPGGVGLGGAGRGGAGRGGAGSVGAGRGGAGRGGTGSEQ ID No. 84: >gi|13561982|gb|AAK30594.1|AF350265_1 flagelliform silk protein[Argiope trifasciata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 ID No. 85: >gi|7106229|gb|AAF36092.1|flagelliform silk protein [Nephila madagascariensis]SGGSGGTTVIEDLDITIDGADGPITISEELTISGAGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGVGPGGTGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGFGPGGSGPGGSGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGYGPGGSGPGGAGPGGAGPGGVGPGGAGPGGAGPGGVGPGGAGPGGAGPGGAGPGGAGRGGAGPGGAGGAGGAGGSGGAGGSGGTTVIEDLDITIEGADGPITISEELTIGGAGGSGPGGAGGSGPGGAGPGGVGPGGSGPGGLGSGGSGPGGVGPGGSGPGGVGPGGYGPGGSGGLYGPGSYGPGGSGVPYGSSGTYGSGGGYGPGGAGGAYGPGSPGGAYGPGSGGSYYPSSRVPDMVNGIMSAMQGSGFNYQMFGNMLSQYSSGSGSCNPNNVNVLMDALLAALHCLSNHGSSSFAPSPTPAAMSAYSNSVGRMFAYSEQ ID No. 86: >gi|13561980|gb|AAK30593.1|AF350264_1 flagelliform silk protein[Argiope trifasciata]AGGPGAGGAGAGGVGPGGFGGPGGFGGAGGPGGPGGPGGAGGGAGGAGGLYGPGGAGGLYGPGGLYGPGGAGVPGAPGASGRAGGIGGAAGGAGAGGVGPGGVSGGAGGAGGSGVTVVESVSVGGAGGPGAGGVGPGGVGPGGVGPGGIYGPGGAGGLYGPGAGGAFGPGGGAGAPGGPGGPGGPGGPGGLGGGVGGAGTGGGVGPGAGGVGPSGGAGGTGPVSVSSTVSVGGAGGPGAGGPGAGGAGAGGVGPGGFGGPGGFGGAGGPGGPGGPGGAGGGAGGAGGLYGFGGAGGLYGPGGLYGPGGAGVPGAPGASGRAGGIGGAAGAGGVGPGGVSGGAGGSGVSVTESVTVGGAGGAGAGGIGGPSGLGGAGATGGFGGRGGPGGPGGPGGPGRFGGAAGGAGAGGVGPGGVSGGAGGAGGSGVTVVESVSVGGAGGPGAGGVGPGGVGPGGVGPGGIYGPGGAGGLYGPGAGGAFGSGGGAGAPGGPGGPGGPGGPGGLGGGVGGAGTGGGVGPGVGGVGPSGGAGGTGPVSVSSTITVGGGQSSGGVLPSTSYAPTTSGYERLPNLINGIKSSMQGGGFNYQNFGNILSQYATGSGTCNYYDINLLMDALLAALHTLNYQGASYVPSYPSPSEMLSYTENVRRYF SEQ ID No. 87: >gi|7106228|gb|AAF36091.1|flagelliform silk protein [Nephila madagascariensis]MGKGRHDTKAKAKAMQVALASSIAELVIAESSGGDVQRKTNVISNALRNALMSTTGSPNEEFVHEVQDLIQMLSQEQINEVDTSGPGQYYRSSSSGGGGGGGGGPVITETLTVTVGGSGAGQPSGAGPSGTGGYAPTGYAPSGSGPGGVRPSASGPSGSGPSGSRPSSSGSSGTRPSANAAGGSSPGGIAPGGSSPGGAGVSGATGGPASSGSYGSGTTGGAYGPGGGSEPFGPGAAGGQYGPGGAGPGGAGAYGPGGVGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGSGTGGAGPGGYTPGGAGPGGYGPGGYGPGGSGPGGAGSGGVGPGGYGPGGAGPGGAGPGGAGPGGAGPSGAGPGGAGTGGAGTGGAGPGGAGPGGAGPGGAGPGGAGRGGAGRGGAGRGGAGRGGAGRGGAGRGGAGGAGGAGGAGGAGGAGGAGGSGSTTIIEDLDITIDGADGPITISEELTIGGAGAGGSGPGGAGPGGVGPGRSGPGGVGPGGSGPGSVGPGGSGQGGLGIGRSGPGGVGPGGSGPGSIGPGGSGQGGLGPGGSGQGGLGPGGSGPGGVGSGGVGGPYGPGGSGPGGVGGAGGPYGPGGSGGPGGAGGPYGPGGPYGPGGAGGPYGPGGAGGPYGPGGPYGPGGAGGPGGGGPGGAGGPYGPGGPGGAGPGGYGPGGYGPGGAGPGGAGPGGYGPGGAGPGGYGPGGAGPGGSGPGGIGPGGSGPGGYGPGGIGPGGTGPGGAGPGGAGPGGAGPSGAGPGGAGPSGAGRGGSGRGSVGRGGAGRGGAGRGGAGGAGGSGGAGGSGGAGGSGGTTIIEDLDITVDGANGPITISEELTIGGAGAGGVGPGGSGPGGVGPGGSGPGGVGPGGSGPGGVGSGGSGPGGVGPGGSGPGGVGSGGFGPGGIGPGGSGPGGVGPGGVGGPYGPGGSGPGGAGGAGGSYGPGGPYGPGGSGGPGGAGGPYGPGGAGGPYGPGGPYGPGGAGGPGGEGPGGAGGPYGPGGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGSGGYGPGGAGPGGYGPGGPGPGGYGPGGAGPGGYGPGGTGPGGSAPGGAGPGGAGPGGYGPGGSGPGGYGPGGGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGVGTGGLGRGGAGRGGAGRGGAGRGGAGRGGAGRGGTGGVGGAGGAGGAGGVGGAGGSGGTTVIEDLDITIDGADGPITISEELTISGAGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYRPGGSGPGGAGGAGGPGGAYGPGGSGGPGGAGGPYGPGGEGPGGSGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGPGGAGGPYGPGGVGPGGTGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGGSGPGGYGPGGSGPGGAGPGGYGPGGTGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGAGPGGAGPGGAGPGGAGPGGVGPGGAGPGGSGPGGAGPGGAGRGGAGRGGAGXGGAGPGGAGGAGGAGGSGGAGGSGGTTVIEDLDITIDGADGPITISEELTINGAGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGPGGAGGPYGPGSEQ ID No. 88: >gi|7106224|gb|AAF36090.1|flagelliform silk protein [Nephila clavipes]AGPSGTGGYAPTGYAPSGSGAGGVRPSASGPSGSGPSGGSRPSSSGPSGTRPSPNGASGSSPGGIAPGGSNSGGAGVSGATGGPASSGSYGPGSTGGTYGPSGGSEPFGPGVAGGPYSPGGAGPGGAGGAYGPGGVGTGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGAGPGGYGPGGTGPGGYGPGGTGPGGVGPGGAGPGGYGPGGAGPGGAGPGGAGPGGAGPGGAGPGGYGPGGSGPGGAGPSGAGLGGAGPGGAGLGGAGPGGAGTSGAGPGGAGPGGAGQGGAGPGGAGRGGAGRGGVGRGGAGRGGAGRGGARGAGGAGGAGGAGGSGGTTIVEDLDITIDGADGPITISEELTIGGAGAGGSGPGGAGPGNVGPGRSGPGGVGPGGSGPGGVGPGSFGPGGVGSGGSGPGGVRPSGSGPGGVGTGGVGPGGAGGPYGPGGSGPGGAGSAGGTYGPGGFGGPGGFGGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGAGGSYGLGGAGGSGGVGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGSGGYGPGGSGPGGSGPGGYGPGGTGPGGSESGGYGPGGSGPGGSGPGGSGPGGSGPGGYGPGGSGPSSFVPGGSGPGGSGPGGAGPGGAGPGGAGPGGAGPGGVGLGGAGRGGAGRGGAGSVGAGRGGAGRGGAGRGGAGRGGAGRGGAGGAGGAGGAGGPGGAGGSGGTTVIEDLDITIDGADGPITISEELTISGAGGSGPGGAGTGGVGPGGSGPGGVGPGGFGPGGVGPGGSGPGGVGPGGAGRPYGPGGSGPGGAGGAGGTGGAYGPGGAYGPGGSGGPGGAGGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGVSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGYGPGGSGPGGSGPGGSGPGGYGPGGTGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAAPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGGAGGAGGSGGAGGSGGTTIIEDLDIT/DGADGPITISEELPISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGGGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGGAGGAGGAGGSGGAGGSGGTTIIEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGSGPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPGGVGPGGGGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGYGPGGSGPGGSGPGGYGPGGSGPGGFGPGGFGPGGSGPGGYGPGGSGPGGAGPGGVGPGGFGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGGAGGAGGAGGSGGAGGSGGTTIIEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGSGAGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYGPGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGPGGAGGPYGPGGAGGPYGPGGEGGPYGPGGSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGPG SEQ ID No. 89: >gi|7106223|gb|AAF36089.1|flagelliform silk protein [Nephila clavipes]VGPGGSGPGGYGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYGSGGAGPGGYGPGGSGPGGYGPGGSGPGGYGPGGTGPGGTGPGGSGPGGYGPGGSGPGGSGPGGSGPGGYGPSGSGPGGYGPSGSGPGGYGPGGSGPGGYGPGGSGAGGTGPGGAGGAGGAGGSGGAGGSGGAGGSGGAGGSGGVGGSGGTTITEDLDITIDGADGPITISEELTISGAGGSGPGGAGPGGVGPGGSGPGGVGPGVSGPGGVGPGGSGPGGVGSGGSGPGGVGPGGYGPGGSGSGGVGPGGYGPGGSGGFYGPGGSEGPYGPSGPYGSGGGYGPGGAGGPYGPGSPGGAYGPGSPGGAYYPSSRVPDMVNGIMSAMQGSGFNYQMFGNMLSQYSSGSGTCNPNNVNVLMDALLAALHCLSNHGSSSFAPSPTPAAMSAYSN aciniform spidroinSEQ ID No. 90: >gi|49871101|gb|AAR83925.1|aciniform spidroin 1 [Argiope trifasciata]SSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSDGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGLLGGGAPFGQSGFGGSDGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSDGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGTGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSDGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQALASSTSGAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAGPQGGFGATGGASAGLISRVANALANTSTLRTVLRTGVSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAVSRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSRVLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLSTSSSSASYSQASASSTSGAGYTGPSGPSTGPSGYPGPLSGGASFGSGQSSFGQTSAFSASGAGQSAGVSVISSLNSPVGLRSASAASRLSQLTSSITNAVGANGVDANSLARSLQSSFSALRSSGMSSSDAKIEVLLETIVGLLQLLSNTQVRGVNPATASSVANSAARSFELVLA tubuliform spidroinSEQ ID No. 91: >gi|63054371|gb|AAY28953.1|tubuliform spidroin 1 [Argiope aurantia]GNAAGLGNALSQAVSSVGVGASSSTYANAVSNAVGQFLAGQGILNXANAGSLASSFASALSASAASVASSAAAQXASQSQAAASAFSRAASQSASQSAARSGAQSSSXTTTTSTSGSQAASQSASSAASQASEQ ID No. 92: >gi|61387231|gb|AAX45291.1|tubuliform spidroin [Argiope aurantia]TTTSTAGSQAASQFASSAASQASASSFARASSASLAASSSFSSAFSSANSLSALGNVGYQLGFNVANNLGIGNAAGLGNALSQAVSSVGVGASSSSYANAVSNAVGQLLAGQGILNAANAGSLASSFASALSASAASVASSAAAQAASQSQAAASAFSRAASQSASQSAARSGAQSISTTTTTSTAGSQAASQSASSAASQASASSFARASSASLAASSSFSSAFSSANSLSALGNVGYQLGFNVANNLGIGNAAGLGNALSQAVSSVGVGASSSTYANAVSNAVGQFLAGQGILNAANAGSLASSFASALSASAASVASSAAAQAASQSQAAASAFSRAASQSASQSAARSGAQSSSTTTTTSTAGSQAASQFASSAASQASASSFARASSASLAASSSFSSAFSSANSLSALGNVGYQLGFNVANNLGISNAAGLGNALSQAVSSVGVGASSSSYANAVSNAVGQFLAGQGILNAANAGSLASSFASALSASAASVASSAAAQAASQSQAAASAFSRAASQSASQSAARSGAQSSSTTTTTSTSEQ ID No. 93: >gi|61387234|gb|AAX45292.1|tubuliform spidroin [Argiope aurantia]STYANAVSNAVGQFLAGQGILNAANAGSLASSFASALSASAASVASSAAAQAASQSQAAASAFSRAASQSASQSAARSGAQSFSTTTTTSTAGSQAASQSASSAASQASASSFARASSASLAASSAFSSAFSSANSLSALGNVAYQLGFNVANTLGIGNAAGLGNALSQAVSSVGVGASSSTYANAVSNAVGQFLAGQGVLNAGNAGSLASSFANALSNSALSVGSRVSSPSYGALSPIAAGPNFISTGLNVGGPFTTLSQSLPTSLQTALAPIVSSSGLGSSAATARVRSLANSIASAISSSGGSLSVPAFLNLLSSVGAQVSSSSSLNSSEVTNEVLLEAIAALLQVINGGSITSVDLRNVPNAQQDLVNALSG SEQ ID No. 94: >gi|61387237|gb|AAX45293.1|tubuliform spidroin [Araneus gemmoides]ASQSQAASQSQAAASAFRQAASQSASQSASRAGSQSSTKTTSTSTSGSQADSRSASSSASQASASAFAQQSSASLSSSSSFSSAFSSATSISAVGNVGYQLGLKVANSLGLGNAQALASSLSQAVSAVGVGASSNAYANAVSNAVGQVLAGQGILNAANAGSLASSFASALSSSAASVASQSASQSQAASQSQAAASAFRQAASQSASQSASRAGSQSSTKTTSTSTSGSQADSRSASSSASQASASAFAQQSSASLSSSSSFSSAFSSATSISAVGNVGYQLGLKVANSLGLGNAQALASSLSQAVSAVGVGASSNAYANAVSNAVGQVLAGQGILNAANAGSLASSFASALSSSAASVASQSASQSQAASQSQAAASAFRQAASQSASQSDSRAGSQSSTKTTSTSTSGSQADSRSASSSASQASASAFAQQSSASLSSSSSFSSAFSSATSISAVGNVGYQLGLKVANSLGLGNAQALASSLSQAVSAVGVGASSNAYANAVSNAVGQVLAGQGILNAANAGSLASSFASALSSSAASVASQSASQSQAASQSQAAASAFRQAASQSASQSASRAGSQSSTKTTSTSTSGSQADSRSASSSASQASASAFAQQSSASLSSSSSFSSAFSSATSISAVGNVGYQLGLKVANS SEQ ID No. 95: gi|61387241|gb|AAX45294.1|tubuliform spidroin [Araneus gemmoides]SASQSQAAASAFRQAASQSASQSASRAGSQSSSKTTSTSTSGSQADSRSASSSASQASASAIAQQSSASLSSSSSFSSAFSSATSLSAVGNVGYQLGLKVANSLGLGNAQALASQGILNAANAGSLASSFASALSASAGSVGNRSSAGPSAVGLGGVSAVPGFISATPVVGGPVTVNGQVLPAALQTALAPVVTSSGLASSAASARVSSLAQSIASAISSSGGTLSVPIFLNLLSSAGAQATASSSLSSSQVTSQVLLEGIAALLQVINGAQIRSVNLANVPNVQQALVSALSG SEQ ID No. 96: >gi|61387244|gb|AAX45295.1|tubuliform spidroin +Nephila clavipes+ASAASSLAYSIGISAARSLGIADAAGLAGALARAAGALGQGDTAASYGNALSTAAGQFFATAGLLNAGNASALASSFARAFSASAESQSFAQSQAFQQASAFQQAASRSASQSAAEADSTSSSTTTTTSAARSQAASQSASSSYSSAFAQAASSSFAISSALSRAFSSVSSASAASSLAYSIGLSAARSLGIADATGLAGALARAVGALGQGATAASYGNALSTAAAQFFATAGLLNAGNASALASSFARAFSASAESQSFAQSQAFQQASAFQQAASRSASQSAAEAGSTSSSTTTTTSAARSQAASQSASSSYSSAFAQAASSSLATSSALSRAFSSVSSASAASSLAYSIGLSAARSLGIADAAGLAGVLARAAGALGQGATAASYGNALSTAAGQFFAAQGLLNAGNVSSLASALANALSYSAANSAASGNYIGVSQNFGSIAPVAGTAGISVGVPGLLPTSAGTVLAPANAQIIAPGLQTTLAPVFSSSGLSSASANARVSSLAQSFASALSASRGTLSVSTFLTLLSPISSQIRANTSLDGTQATVQVLLEALAALLQVINAAQITEVNVSNVSSANAALVSALAG

EXAMPLES Example 1 Engineering of Recombinant Spider Silk ProteineADF4(C16)

The amino acid sequence of eADF4 (C16) was adapted from the naturalsequence of ADF4 from Araneus diadematus. eADF4(C16) protein wasengineered by the combination and multimerization of single motifs. Theresulting eADF4(C16) comprises 16 repeats of Modul C with the amino acidsequence GSSAAAAAAA ASGPGGYGPE NQGPSGPGGY GPGGP (SEQ ID NO: 21). Theresulting protein has a molecular mass of 48 kDa. The protein waspurified as described previously (Hümmerich et al., 2004) having apurity higher than 98%. Due to its amino acid composition, eADF4(C16)has a theoretical isoelectric point of 3.48 indicating a net negativecharge at a physiological pH of 7.4.

Example 2 Preparation of Small Molecular Model Drugs

All drugs were dissolved in water at a concentration of 0.21 μmol/ml.Drug substances and their featured properties are depicted in Table 1.The main selection criteria were solubility in aqueous media (expressedby the octanol/water partition coefficient (log P)), the acidicdissociation constant (pKa for protonated bases (BH+) or for acids (HA))and the resulting net-charge in aqueous media (predominant or permanentcharge).

TABLE 1 List of small molecular weight model drugs used for eADF4(C16)sphere loading. Values for molecular weight, dissociations constants(pKa) and partition coefficients (logP) are taken from literature. Thepartition coefficient (logP) accounts for the individual unprotonatedforms. The absorption wavelength λAbs was determined experimentally foreach substance. All substances were purchased from Sigma-Aldrich(Deisenhofen, Germany). Molecular Dissociation Dissociation Predominantweight λ_(Abs) constant of constant of charge at Permanently Model drug[Da] (nm) BH⁺(pKa) HA (pKa) log P pH7 charged Phenol red 354 510 — 1.7;7.7 3.00 negative yes Tetracaine*HCl 301 310 8.20 — 4.00 positive noProcaine*HCl 272 290 8.05 — 2.40 positive no Papaverine*HCl 376 248 8.07— 3.50 positive no Ephedrine*HCl 202 256 9.60 — 1.30 positive noPropranolol*HCl 295 290 9.10 — 3.18 positive no Ethacridine lactate 343365 11.00  — 2.50 positive no Methyl violet 407 590 — — 3.20 positiveyes

Example 3 Preparation of eADF4(C16) Particles

Lyophilized protein eADF4(C16) was dissolved in 6 Mguanidiniumthiocyanate. Dialysis was performed against 10 mMtris(hydroxymethyl)aminomethane-(Tris)/HCl, pH 8, at 4° C. usingmembranes with a molecular weight cut-off at 6000-8000 Da (SpectrumLaboratories, Rancho Dominguez, USA). The concentration of eADF4(C16)solution was determined by UV-Vis-spectrometry at 20° C. using a Cary100spectrophotometer (Varian Medical Systems, Palo Alto, USA) and the molarextinction coefficient of eADF4(C16) at 276 nm (ε=46400 M-1 cm-1). eADF4(C16) particles were prepared using a phase separation procedure asdescribed previously in Slotta et al. (2008). An aqueous eADF4(C16)(c=1.0 mg/ml) solution was mixed with potassium phosphate (2 M, pH 8) involumetric ratios of 1:10 using a pipette. The resulting particles werecentrifuged for 10 min at 10.000×g and washed three times with purifiedwater. The obtained particles were redispersed in water, and particleconcentrations (particles in mg/ml) were determined gravimetrically. Astock dispersion of known protein particle concentration was used forall experiments.

Example 4 Colloidal Stability of eADF4(C16) Particles

The colloidal stability of eADF4(C16) particles in suspension wasstudied by adding 1.0 mg of particles to 1.0 ml of (NH₄)₂SO₄ solutionsof varying concentration (0-2.0 M) and measuring the intensity ofscattered light at a wavelength of 400 nm after 15 min. Based on the Mietheory, the intensity of scattered light in forward direction increaseswith increasing particle sizes. Therefore, the onset ofelectrolyte-induced flocculation in dilute dispersions can be detectedby an increase in intensity of scattered light in forward direction.

Example 5 Characterization of eADF4(C16) Particles

The following methods of the state of art can be used to characterizethe spider silk particles according to the invention:

a) Scanning Electron Microscopy

The eADF4(C16) particles were immobilized on Thermanox plastic coverslips (Nagle Nunc, USA), dried at room temperature, gold sputtered undervacuum, and analyzed with a JSM 5900 LV scanning electron microscope(JEOL Ltd., Japan, at 20 kV).

b) Laser Diffraction Spectrometry

Particle sizes and size distributions were determined in triplicateusing laser diffraction spectrometry (Horiba, Partica LA-950, Japan).Refractive indices of 1.33 for water and 1.60 for protein were taken forcomputation of particle sizes. In order to eliminate concentrationeffects, all samples were measured at equal concentrations resulting ina transmittance of 82%. In addition, a dry specimen of each preparationwas analyzed by scanning electron microscopy (SEM) to confirm sphericalshape and sphere sizes.

c) Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transform infrared (FTIR) spectra were collected using a BrukerEquinox 55 FTIR spectrometer. The samples were prepared by putting adroplet of eADF4(C16) particle suspension on CaF2 disks and subsequentair-drying. Absorbance spectra were recorded between 400 and 4000 cm-1with unpolarized light at a resolution of 4 cm⁻¹. The measurements werecarried out at 25° C. and 30% relative humidity and each spectrum wasaccumulated 32 times. The secondary structure of eADF4(C16) particleswas analyzed using the amide I band (1600-1700 cm-1). Peaks at 1648-1660cm⁻¹, 1625-1640 cm⁻¹ and 1660-1668 cm⁻¹ can be assigned to α-helical,β-sheet and β-turn structures, respectively

d) UV-Vis-spectroscopy

Ultraviolet-visible spectrometry, using a Cary100 spectrophotometer(Varian Medical Systems, Palo Alto, USA), has been employed fordetermination of the drug concentration in supernatants as a basis forthe calculation of loading efficiencies and release behaviour.Calibration curves for all model drugs have been obtained by using fivedifferent concentrations of all stock solutions.

e) Zetapotential Analysis

In order to elucidate and characterize the loading mechanism ofeADF4(C16) particles with model drugs, zeta potential measurements wereconducted as a function of amount of model drug added. The zetapotential was determined using a Nanoseries Malvern Zetasizer (Malvern,Worcestershire, UK). Automatic titration was conducted with a MalvernMultipurpose Titrator MPT-2. Experiments were performed in distilledwater (pH 7) at 25° C. Each measurement was performed in triplicate.

To characterize the morphology and determine the sizes of obtainedeADF4(C16) particles, the prepared stock dispersion was examined usingSEM and laser diffraction spectrometry. As shown in FIG. 1 a), particlesof spherical shape with diameters from 170 nm to 700 nm were obtained.The determined average diameter of particles was d_(avg)=332±95 nm. Theyield of particle formation by salting-out was higher than 99% withremaining soluble protein below the detection limit. It could beobserved that eADF4(C16) particles are colloidally stable within thecomplete studied concentration range from 0 to 2.0 M (NH₄)₂SO₄ (FIG. 1b). The slight linear decrease of intensity with increasingconcentration of (NH₄)₂SO₄ can be explained by the linear increase inion concentration yielding a decrease of number of particles per volume.

Example 6 Drug Loading of eADF4(C16) Particles

Drug loading of spider silk particles was conducted as follows: 100 μlof spider silk particle suspension containing 21 nmol silk protein weremixed with 1.0 ml of model drug solution containing 0.21 μmol modeldrug. After 10 min of incubation at room temperature samples werecentrifuged for 10 min at 10.000 g, and the supernatant was analyzed forresidual drug concentration using UV-Vis spectrometry. Standardcalibration curves for model drugs were used for drug quantification. Acontrol group of samples containing only 100 μl water mixed with 1.0 mlof model drug solution was prepared for each experiment. Drugconcentrations from control and sample supernatants were used tocalculate the amount of drug incorporated in the spider silk particles.All experiments were performed in triplicate. Encapsulation efficiencyand loading were determined by using equation (1) and (2), respectively:

$\begin{matrix}{{{encapsulation}\mspace{14mu}{efficiency}\mspace{14mu}\left( {w\text{/}w\mspace{14mu}\%} \right)} = {\frac{{amount}\mspace{14mu}{of}\mspace{14mu}{model}\mspace{14mu}{drug}\mspace{14mu}{in}\mspace{14mu}{particles}}{{model}\mspace{14mu}{drug}\mspace{14mu}{initially}\mspace{14mu}{added}} \times 100}} & (1) \\{\mspace{20mu}{{{loading}\mspace{14mu}\left( {w\text{/}w\mspace{14mu}\%} \right)} = {\frac{{amount}\mspace{14mu}{of}\mspace{14mu}{model}\mspace{14mu}{drug}\mspace{14mu}{in}\mspace{14mu}{particles}}{{amount}\mspace{14mu}{of}\mspace{14mu}{particles}} \times 100}}} & (2)\end{matrix}$

Example 7 Loading Efficiencies and Loading Procedure

Due to its negative charge at pH 7, eADF4(C16) can form complexes withpositively charged molecules based on electrostatic interactions. Inorder to elucidate if small molecules attach to the particle surface orare able to permeate into the interior, loading efficiencies of glassbeads were compared with that of eADF4(C16) particles assuming thatpermeation processes of drug molecules into the dense glass matrixcannot occur. Due to the high negative zeta potential (≈−50 mV) of glassbeads, the loading efficiency of glass beads should be higher than thatof spider silk particles (zeta potential≈−22 mV) if no diffusion intothe protein matrix occurs.

For this experiment methyl violet (MV) was employed with loadingefficiencies above 95% at molar ratios of MV:eADF4(C16) of 10:1. Onlinezeta potential measurements during methyl violet loading revealed thatthe change of zeta potential during eADF4(C16) particle loading is atriphasic process (FIG. 2 a). First, the potential changes graduallyafter addition of methyl violet solution. After an initial constantslope, the zeta potential curve exhibits a plateau phase, indicating nofurther change of surface loading upon increasing methyl violetconcentration. Finally the zeta potential decreases further. Thereduction of the zeta potential, as seen in the titration curve, is adirect consequence of the interaction of the silk particles withmolecules of opposite charge. The initial lowering of surface charge canbe explained by the charge compensation due to the addition of oppositecharged methyl violet molecules. The plateau region indicates anequilibrium state of drug (compound) adsorbed at the solid-liquidparticle interface and a diffusion of molecules into the hydrophobiccore of the protein sphere. Said second phase is mainly characterized bythe diffusion of the drug (compound) into the matrix of the particle,whereas the first phase is mainly characterized by the adsorption of thedrug (compound) to the surface of the particle. After the core matrix issaturated, the influx of methyl violet molecules is reduced andeventually terminated. At that point the zeta potential starts todecrease again, as can be seen by the second slope in FIG. 2 a, due tofurther loading of the particle surface. The decrease occurs at a methylviolet concentration corresponding to the molar ratio of MV:eADF4(C16)of 10:1 which was identified to be the molar ratio at which the loadingefficiency decreases. FIG. 2 b shows the obtained loading and loadingefficiencies employing eADF4(C16) particles as a function of molarratio. Up to a molar ratio of MV:eADF4(C16)≈10 the loading increaseslinearly with the amount of methyl violet added. Above a molar ratio of10 the loading reaches a plateau leading to a decrease of loadingefficiency.

In contrast, the zeta potential of glass microparticles during methylviolet addition showed no distinctive changes (inset FIG. 2 a). Theinitial assumption that methyl violet cannot permeate the glass particlematrix was confirmed by analyzing the supernatant after completing thetitration experiment. While the surface charge of glass particles isapproximately two times higher compared to silk particles, thedetermined loading efficiency was only 0.03%. Furthermore, the loadedmethyl violet could be easily washed off the surface of glass particlesby three washing steps using Millipore water.

In order to investigate the influence of molecular parameters on theloading efficiency, twelve different small molecular drugs were used inthis study (see Table 1). Since an individual eADF4(C16) molecule isamphiphilic with a dominating hydrophobic character (hydropathicityindex=−0.46) exhibiting 17 negative charges (one at each C module andone at the carboxy terminus) and one positive charge at the aminoterminus, it can be concluded that loading of eADF4(C16) particles withdrugs is mainly driven by three parameters: (i) the charge of the drugmolecule determined by its proton dissociation constant Ka (accountedfor BH+ or HA), (ii) the octanol water partition coefficient (logP_(o/w)), as an indicator of solubility of the model drug, and (iii) themolecular weight (MW) which plays an important role in diffusion drivenmass transport processes.

Further, the distribution between a hydrophobic and a hydrophilic phaseof two different species of a specific drug, i.e. the native and theprotonated form, can be described by its apparent distributioncoefficient (log D), which can be calculated with equations (3) and (4)respectively.for acids: log D=log P−log(1+10^(pH-pKa))  (3)for bases: log D=log P−log(1+10^(pKa-pH))  (4)

The log P and pKa values of individual species used for calculation oflog D are listed in Table 1. Table 2 summarizes the determined loadingefficiencies, maximal (calculated by employing loading efficiencies of100%) and experimental amount of entrapped drug, as well as thecalculated distribution coefficient (log D) at pH 7.

TABLE 2 List of employed model drugs classified according to theirchemical nature. The table provides an overview of theoretical andexperimental model drug content of loaded spider silk particles(expressed as percentage of wt drug/wt spider silk protein particles),corresponding encapsulation efficiencies and calculated distributioncoefficients (logD). Experi- Encap- Maximal mental sulation drug drugeffi- Chemical content content ciency Model drug nature [w/w %] [w/w %][%] log D Ephedrin•HCl base 4.23 0.88 20.7 −1.321 Frocain•HCl base 5.712.16 38.0 0.396 Propranolol•HCl base 6.19 2.78 45.0 1.197 Papaverine•HClbase 7.89 3.71 47.0 2.395 Tetracaine•HCl base 6.30 3.34 53.0 2.773Ethacridine strong 7.20 7.07 98.2 2.899 lactat base Phenol red strong7.12 0.00 0.0 — acid Methyl violet — 8.54 8.37 98.1 —

Protonated weak organic bases were able to be loaded onto eADF4(C16)particles with efficiencies ranging between 20.7% and 53.0%. For thisclass of small molecular model drugs the quotient of calculated log Ddivided by the molecular mass of the individual molecule correlateslinearly with the obtained loading efficiencies (see FIG. 3). Thislinear relationship clearly indicates that the combination of charge andsolubility (expressed by the apparent distribution coefficient log D)and diffusion coefficient (expressed by the inverse proportionality ofmolecular weight) are the dominating factors responsible for effectiveloading of small weakly alkaline molecules onto eADF4(C16) particles.

Investigation of molecules with permanent charge revealed thatpositively charged molecules such as methyl violet were mostsuccessfully incorporated, whereas negatively charged molecules such asphenol red could not be incorporated using eADF4(C16), and slightlyacidic molecules exhibited relatively low loading efficiencies from 0.2to 17.3%. Strongly alkaline molecules such as ethacridine lactate showeda loading efficiency of more than 98%.

Example 8 In vitro Release Studies

Drug loaded eADF4(C16) particles were washed with distilled water andsuspended in 1 ml PBS (pH 7.4) before incubation at 37° C. with constantshaking. Each vial contained 2 mg of drug loaded particles containing4.2 μmol spider silk protein. The solvent was periodically removed fromeach sample and replaced with fresh PBS (pH 7.4). The drug content inthe medium was then analyzed using UV-Vis-spectrometry. The percentageof cumulative model drug release (% w/w) was investigated as a functionof incubation time. Each experiment was performed in triplicate. Tostudy the effect of different pH values on the release behaviour of drugloaded eADF4(C16) particles, 1 mg drug loaded silk particles wereincubated in 1.0 ml PBS at 5 different pH values (pH 2, 4, 6, 7.4 and8.8) for 5 days. The solvent was withdrawn daily and the particles wereredispersed in fresh media. Supernatants of drawn samples were analyzedfor drug content determination with UV-Vis-spectrometry.

The in vitro release behavior of model drugs from eADF4(C16) particleswas exemplarily studied with methyl violet and ethacridine lactate.Cumulative release profiles showed that both molecules were releasedover a period of 30 days (FIG. 4 a). Most interestingly, only a verysmall drug burst could be detected, i.e. an initial higher drug releasewithin the first 24 hours of incubation. The release of ethacridinelactate and methyl violet within the first 24 hours was 11% of the totalamount encapsulated. Subsequently, eADF4(C16) particles releasedapproximately 5% of the entrapped molecules per day within the firstweek (FIG. 4 a,b). To characterize the release behavior, the semiempirical power law equation was used (equation (5)),

$\begin{matrix}{\frac{M_{t}}{M_{\infty}} = {kt}^{n}} & (5)\end{matrix}$where Mt/M∞ is the fractional amount of the drug released at time t, kis a characteristic constant of the system, and exponent n is related tothe geometrical shape of the formulation and is indicative of themechanism of drug release. The semi-empirical power law can be seen as ageneralization of two independent mechanisms of drug transport, Fickiandiffusion and Case II transport, reflecting the influence of polymerrelaxation on molecules' movement in the matrix. For spherical systemsthe limiting value of n, when pure Fickian diffusion or pure Case IItransport is operating, were determined to be equal to 0.43 and 0.85,respectively [42]. When n is between 0.43 and 0.85, a superposition ofboth transport processes occurs which is known as anomalous transport.In order to obtain a linear fit for the drug release data, equation (5)was modified leading to equation (6),

$\begin{matrix}{{\log\left( \frac{M_{t}}{M_{\infty}} \right)} = {{\log(k)} + {n\;{\log(t)}}}} & (6)\end{matrix}$where n can be calculated from the slope of the log-log plot of releaseMt/M∞ versus time t by linear fitting (FIG. 4 d). Therefrom, three timeintervals with different dominating release mechanisms could beidentified excluding the initial burst region (<24 h). To distinguishbetween different time intervals, the criterion that the coefficient r2had to be above 0.99 for the individual linear fits was employed. Thevalues of release exponent (n), correlation coefficient (r2), andcharacteristic constant (k) are summarized in Table 3. For validation ofthe determined release parameters, the experimental release data werecompared with theoretical data obtained by the semi-empirical power lawemploying the determined values for k and n. A very good agreement frompost-initial burst stage (>24 hours) up to 100% release was obtained(FIG. 4 a). Since only release data after 24 hours were considered forcalculation of release parameters (k and n), the initial burst isunderestimated by theoretical data (FIG. 4 b).

TABLE 3 Drug release parameters (n: release exponent; r²: correlationcoefficient; k: characteristic constant) for methyl violet andethacridine lactate for defined release intervals. Model drug time [d]Release [%] n r² k Methyl violet  0-13 ≦60 0.692 0.998 1.17 14-20 60-820.6079 0.994 1.92 >20 ≧82 0.3537 0.993 9.20 Ethacridine lactate  0-13≦60 00.6754 0.998 1.25 14-20 60-73 0.5083 0.994 3.18 >20 ≧73 0.26410.992 14.4

Within the first two weeks of release, the exponents n for ethacridinelactate (EL) and methyl (MV) violet are almost identical (nEL=0.6754,nMV=0.692), indicating an anomalous diffusional release. In the secondtime interval between day 14 and day 20, release profiles diverge fromeach other with the release exponent of ethacridine lactate dropping to0.51 and that of methyl violet to n=0.61. In this second time interval,fickian transport begins to dominate for ethacridine lactate. After 20days, release exponent n values for methyl violet and ethacridinelactate fall below the limiting value of n=0.43 indicating a fickianrelease behaviour for both (Table 3).

Next, the influence of pH on drug release was evaluated. Releaseexperiments with ethacridine lactate loaded eADF4(C16) particlesincubated in PBS at 37° C. and different pH values showed a strong pHinfluence on the release rates (FIG. 4 c) with an acidic environmentaccelerating drug release. Almost 80% of the loaded drug was releasedafter 24 hours from silk spheres incubated at pH 2 (non bufferedconditions). For silk particles incubated at pH 4 (non-bufferedconditions) an initial release rate of almost 40% was obtained after thefirst day of incubation. Particles incubated at pH 6 showed double therelease with a similar release profile as seen at pH 7.4 or 8.8, whichwere indistinguishable. The observed results confirm the predictedimportance of electrostatic interactions between eADF4(C16) and drugmolecules. Presumably an influx of protons into the biopolymer leads toa displacement of drug molecules from the matrix. In addition, thedecreased pH influences the distribution of charged drug species byshifting the equilibrium towards the charged species. As these speciesare driven towards the negatively charged surface of the protein, theycan easily be washed away by the solvent.

Example 9 In vitro Degradation of eADF4(C16) Particles

In order to analyze, the degradability of eADF4(C16) silk particles, amixture of elastase and trypsin (both naturally occurring proteases invertebrates) were used. 1.0 mg of silk particles was incubated in 1.0 mlPBS in the presence of 0.8 μg elastase and 12.5 μg trypsin. Over twoweeks samples were drawn on a daily basis and centrifuged. The pelletscontaining eADF4(C16) particles were redispersed in distilled water andwashed three times for further analysis of size and morphology usinglaser diffraction spectrometry and scanning electron microscopy.Elastase and trypsin from hog pancreas were supplied by Sigma Aldrich(St. Louis, USA).

Degradability of drug depot systems is a highly desirable property,since the risk of inflammation and intoxication is dramatically lowerthan for non-degradable systems. As most biopolymers feature the abilityof enzymatic degradation, degradation studies were conducted usingproteases (trypsin and elastase) naturally occurring in vertebrates tosimulate a native-like degradation of eADF4(C16) drug carriers. Elastaseand trypsin, i.e. serine proteases, can cleave peptide bonds on thecarboxy side of small, hydrophobic amino acids such as glycine, alanine,and valine. Due to the relative high content of glycine and alanine ineADF4(C16) (≈50% of the total amino acid composition) such proteases maycleave peptide bonds at several sites in the amino acid backbone ofeADF4(C16).

Size and morphology analysis of particle ensembles drawn fromdegradation experiments using LDS and SEM showed that after two days ofdegradation particles form clusters (FIG. 5 a). By comparing the modevalue, which represents the particle size most commonly found in thedistribution, with the mean size of the particles leads to theconclusion that bigger particles of the ensemble are degradedpreferentially (FIG. 5 b).

At t=0 the mean is larger than the mode, indicating an asymmetric sizedistribution towards larger particles. Upon enzymatic degradation fortwo days mean and mode approach each other, indicating that largerparticles disappear and the particle distribution becomes symmetric. Theparticle distribution remains symmetrical up to day 8 at which timepointthe mean falls below the mode, indicating an asymmetric sizedistribution towards smaller particles.

Analysis of the relative relation of single particles to agglomerationsindicates a oscillatory agglomerative behaviour (FIG. 5 a, c).

Changes of secondary structure of eADF(C16) particles can be mosteffectively detected by 2nd derivative changes of FTIR spectra at thewave numbers 1648-1660 cm-1 and 1625-1640 cm-1. The results indicate(FIG. 5 d) that only minor changes in percental β-sheet and α-helicalcontent occur. The overall structure of eADF4(C16) particles isconserved. This is an important result regarding the long term stabilityand release behaviour of eADF4(C16) particles at physiologicalconditions, since structural changes would significantly alter therelease properties.

Example 10 Protein Loading and in vitro Degradation of C₁₆ Spider SilkParticles

The following protein compounds were chosen for loading experiments:

-   (a) Lysozyme, a protein compound with an isoelectric point of 11.35.    The protein exhibits an overall positive net charge at the    investigated pH of 7.0 and has a molecular weight of 14.3 kDa.-   (b) Nerve growth factor (NGF) has an isoelectric point of 9.5 and a    molecular weight of 13 kDa. NGF it is also positively charged at the    investigated pH of 7.0.

Loading with lysozyme was conducted in buffer (10 mM phosphate, pH 7.0)at different ionic strengths of 30 mM, 60 mM and 100 mM (adjusted withsodium chloride). The loading procedure as applied in example 6 wasmodulated and implemented as follows: A stock dispersion of spider silkparticles was centrifuged and redispersed in the desired buffer mediabefore loading. A second stock solution comprising lysozyme was preparedby dissolving lyophilized lysozyme in an identical buffer solution.Spider silk particle suspension and lysozyme stock solution were mixedto achieve a final spider silk particle concentration of 0.5 mg/ml.After 30 minutes of incubation at room temperature under gentleagitation, 20 μl of the resulting particle suspension were used fordynamic light scattering measurements. Simultaneously, samples werecentrifuged and the supernatant was analyzed for residual proteincontent using the Micro BCA Protein Assay Kit (Thermo Scientific).Encapsulation efficiencies and loading were determined according toexample 6 by using equation (1) and (2), respectively.

Lysozyme was loaded onto C₁₆ spider (eADF4 (C16)) silk particles in highamounts (see FIG. 6A). At an ionic strength of 30 mM it was possible toload more than 30% [w/w] lysozyme onto the spider silk particles. Theassociated loading efficiencies remain >90% up to 30% w/w-ratios rangingfrom 6 to 20%, representing a very effective loading of lysozyme. Itcould be shown that loading of particles with lysozyme did not show asignificant change in the zeta-potential of particles up to loading of30% (see FIG. 6B). This argues that lysozyme diffuses into the matrixand is not mainly adsorbed to the particles' surface.

The loading of a particle with lysozyme does not result in a significantincrease of the zeta potential, which corresponds to no detectableincrease of the median of the spider silk particle. Therefore, it can beconcluded that the compound (lysozyme) permeates/diffuses into thespider silk particle.

According to a model calculation with 250 μg of almost 10% [w/w] loadedspider silk particles, a maximum of only 12.5% of the totally loadedcompound (lysozyme with a hydrodynamic diameter of 4.1 nm) could betheoretically located as a monolayer on the surface of the particle.

For calculation the closest/densest sphere packing of lysozyme on thespider silk particle is taken. In contrast to permeation into the spidersilk particle the adsorption of lysozyme molecules at the surface wouldincrease the diameter of the particle for about 80 nm. Surprisingly, noincrease of z-average and thus no increase of particle-size could bedetected. This further argues for the permeation/diffusion of lysozymemolecules into the matrix of the particle. FIG. 8 shows no increase insize of the (eADF4) C₁₆ spider silk particles loaded with approximately10% [w/w] lysozyme compared to unloaded (eADF4) C₁₆ spider silkparticles.

The loading efficiency ranges above 90% for w/w ratios up to 30%,representing a very effective loading process (more than 90% of theoverall added lysozyme is bound to/permeated into the particle). At w/wrations above 30% the loading efficiency slowly decreases, resulting inhigher amounts of unloaded lysozyme in solution.

FIG. 7 displays the influence of ionic strength on the loading oflysozyme into spider silk particles. An increase of ionic strength from30 to 100 mM leads to a distinct decrease in loading and loadingefficiencies. For example, loading at 30% w/w-ratio is reduced from 28%at 30 mM to 24% at 60 mM and 20% at 100 mM.

The invention claimed is:
 1. A method of producing spider silk particlesloaded with a compound comprising the steps of: i) providing spider silkparticles that consist of an inner solid matrix with an outer surface,both the inner solid matrix and the outer surface homogenouslycomprising one or more spider silk polypeptides, wherein the one or morespider silk polypeptides comprise at least two identical repetitiveunits, and wherein the spider silk particles are produced by proteinaggregation, and ii) incubating said spider silk particles with at leastone compound.
 2. The method of claim 1, wherein the spider silkparticles provided in step i) are produced by the steps of: a) providingan aqueous solution comprising one or more spider silk polypeptidescomprising at least two identical repetitive units, b) triggeringaggregation of the spider silk polypeptides to form spider silkparticles, and c) separating the spider silk particles by phaseseparation.
 3. The method of claim 1, wherein the compound is able topermeate into the spider silk particles.
 4. The method of claim 1,wherein at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the loadedcompound is located within the spider silk particles.
 5. The method ofclaim 1, wherein the at least two identical repetitive units eachcomprise at least one consensus sequence selected from the groupconsisting of: i) GPGXX (SEQ ID NO:3), wherein X is any amino acid,preferably in each case independently selected from the group consistingof A, S, G, Y, P and Q; ii) GGX, wherein X is any amino acid, preferablyin each case independently selected from the group consisting of Y, P,R, S, A, T, N and Q; and iii) A_(x), wherein x is an integer from 5 to10.
 6. The method of claim 5, wherein the repetitive unit of therespective spider silk polypeptide is independently selected from moduleA (SEQ ID NO:20) or variants thereof, module C (SEQ ID NO:21) orvariants thereof, module Q (SEQ ID NO:22) or variants thereof, moduleA^(C) (SEQ ID NO:29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ IDNO:31), module C^(K1) (SEQ ID NO:32), module C^(K2) (SEQ ID NO:33) ormodule C^(KC) (SEQ ID NO:34).
 7. The method of claim 6, wherein thespider silk polypeptide further comprises at least one non-repetitive(NR) unit.
 8. The method of claim 7, wherein the non-repetitive (NR)unit is independently selected from the group consisting of NR3 (SEQ IDNO:41 and SEQ ID NO:45) or variants thereof and NR4 (SEQ ID NO:42 andSEQ ID NO:46) or variants thereof.
 9. The method of claim 1, wherein thecompound is a pharmaceutically active compound, a cosmetic substance, anagricultural substance, a chemoattractant, a chemorepellent, ananti-fungal substance, an anti-bacterial substance, a nutrient, adietary supplement, a dye, a fragrance or an agent selected from thegroup consisting of hemostatic agents, growth stimulating agents,inflammatory agents, anti-fouling agents, antimicrobial agents and UVprotecting agents.
 10. The method of claim 1, wherein the compound hasan overall positive net charge.
 11. The method of claim 1, wherein thecompound is able to permeate into the spider silk particles byelectrostatic interaction and/or diffusion.
 12. The method of claim 1,wherein the compound has a neutral or alkaline nature.
 13. Spider silkparticles produced from the method of claim 1, which consist of an innersolid matrix with an outer surface, both the inner solid matrix and theouter surface homogenously comprising at least one spider silkpolypeptide comprising at least two identical repetitive units, whereinthe spider silk particles are loaded with at least one compound.
 14. Thespider silk particles of claim 13, wherein, at least 40%, 50%, 60%, 70%,80%, 90%, or 95% of the loaded compound is located within the spidersilk particles.
 15. The spider silk particles of claim 13, wherein thespider silk polypeptide comprises at least two identical repetitiveunits each comprise at least one consensus sequence selected from thegroup consisting of: i) GPGXX (SEQ ID NO:3), wherein X is any aminoacid, preferably in each case independently selected from the groupconsisting of A, S, G, Y, P and Q; ii) GGX, wherein X is any amino acid,preferably in each case independently selected from the group consistingof Y, P, R, S, A, T, N and Q; and iii) A_(x), wherein x is an integerfrom 5 to
 10. 16. The spider silk particles of claim 15, wherein therepetitive unit of the spider silk polypeptide is independently selectedfrom module A (SEQ ID NO:20) or variants thereof, module C (SEQ IDNO:21) or variants thereof, module Q (SEQ ID NO:22) or variants thereof,module A^(C) (SEQ ID NO:29), module A^(K) (SEQ ID NO:30), module C^(C)(SEQ ID NO:31), module C^(K1) (SEQ ID NO:32), module C^(K2) (SEQ IDNO:33) or module C^(KC) (SEQ ID NO:34).
 17. The spider silk particles ofclaim 16, wherein the spider silk polypeptide further comprises one ormore non-repetitive (NR) units.
 18. The spider silk particles of claim17, wherein the NR unit is independently selected from the groupconsisting of NR3 (SEQ ID NO:41 and SEQ ID NO:45) or variants thereofand NR4 (SEQ ID NO:42 and SEQ ID NO:46) or variants thereof.
 19. Thespider silk particles of claim 13, wherein the compound is apharmaceutically active compound, a cosmetic substance, an agriculturalsubstance, a chemoattractant, a chemorepellent, an anti-fungalsubstance, an anti-bacterial substance, a nutrient, a dietarysupplement, a dye, a fragrance or an agent selected from the groupconsisting of hemostatic agents, growth stimulating agents, inflammatoryagents, anti-fouling agents, antimicrobial agents and UV protectingagents.
 20. The spider silk particles of claim 13, wherein the compoundhas an overall positive net charge.
 21. The spider silk particles ofclaim 13, wherein the compound is able to permeate into the spider silkparticles by electrostatic interaction and/or diffusion.
 22. The spidersilk particles of claim 13, wherein the compound has a neutral oralkaline nature.
 23. The spider silk particles of claim 13, wherein thecompound is released from the spider silk particles by diffusion uponexposure to physiological conditions.
 24. The spider silk particles ofclaim 23, wherein less than 20%, preferably less than 15%, and mostpreferably less than 10% of the compound is released within the first 24hours.
 25. A pharmaceutical composition comprising the spider silkparticles according to claim 19, and additionally a pharmaceuticallyacceptable buffer, diluent and/or excipient, the pharmaceuticalcomposition being useful for controlled and sustained delivery, whereinthe compound is a pharmaceutically active compound.
 26. A cosmeticcomposition comprising the spider silk particles according to claim 19for controlled and sustained delivery, wherein the compound is acosmetic compound.